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Sample records for affinities ki values

  1. Identification of Inhibitor Concentrations to Efficiently Screen and Measure Inhibition Ki Values against Solute Carrier Transporters

    PubMed Central

    Zheng, Xiaowan; Polli, James

    2010-01-01

    The objective was to identify inhibitor concentrations to efficiently screen and measure inhibition Ki values of solute carrier (SLC) transporters. The intestinal bile acid transporter and its native substrate taurocholate were used as a model system. Inhibition experiments were conducted using 27 compounds. For each compound, the inhibition constant Ki was obtained from the comprehensive inhibition profile, and referred as the reference Ki. Ki values were also estimated from various partial profiles and were compared to the reference Ki. A screening Ki was estimated from one data point and also compared to the reference Ki. Results indicate that Ki can be accurately measured using an inhibitor concentration range of only 0-Ki via five different inhibitor concentrations. Additionally, a screening concentration of 10-fold the substrate affinity Kt for potent inhibitors (Ki < 20Kt) and 100-fold Kt for nonpotent inhibitors (Ki > 20Kt) provided an accurate Ki estimation. Results were validated through inhibition studies of two other SLC transporters. In conclusion, experimental conditions to screen and measure accurate transporter inhibition constant Ki are suggested where a low range of inhibitor concentrations can be used. This approach is advantageous in that minimal compound is needed to perform studies and accommodates compounds with low aqueous solubility. PMID:20553862

  2. Prognostic value of Ki-67 expression in conversion therapy for colorectal liver-limited metastases

    PubMed Central

    Hayashi, Hiromitsu; Beppu, Toru; Sakamoto, Yasuo; Miyamoto, Yuji; Yokoyama, Naomi; Higashi, Takaaki; Nitta, Hidetoshi; Hashimoto, Daisuke; Chikamoto, Akira; Baba, Hideo

    2015-01-01

    Introduction: The objective of this study was to examine the prognostic value of Ki-67 expression in conversion therapy for colorectal liver-confined metastases. Methods: We enrolled a total of 96 patients including 54 patients who received oxaliplatin- or irinotecan-based chemotherapy and curative hepatectomy for initially unresectable metastases (conversion group) and 42 patients with initially resectable liver metastases (straight hepatectomy group). Ki-67 expression was examined in 96 resected specimens but excluded the 2 specimens that revealed no residual cancer cells in conversion group. Results: Conversion therapy leads to greater survival that is equivalent to that straight hepatectomy group. In conversion group, high Ki-67 expression (> 30%) levels were detectable in 33 patients (64%) after chemotherapy prior to conversion therapy. High Ki-67 expression was significantly associated with shorter disease-free survival and worse overall survival (P < 0.01 in both), and was an independent worse prognostic factor of disease-free survival and overall survival (hazard ratio [HR] and P-values were 5.608, 0.001 and 5.366, 0.04, respectively) in patients with conversion therapy. Interestingly, even in the patients with RECIST PR (n = 32), high Ki-67 expression was significantly shorter disease-free survival compared to low Ki-67 expression (P < 0.001). In contrast to conversion group, there was no significant difference in disease free survival and overall survival between low (n = 14, 33%) and high (n = 28, 67%) Ki-67 expressions in patients with straight hepatectomy (P = 0.14 and 0.74, respectively). Conclusions: Residual Ki-67 expression is a useful biomarker for worse prognostic outcomes after conversion therapy. High Ki-67 expression may be a biomarker of micrometastases containing aggressive cancer cells. PMID:26046001

  3. The Reliability of Estimating Ki Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC50 Values: A Retrospective Analysis of 343 Experiments.

    PubMed

    Haupt, Lois J; Kazmi, Faraz; Ogilvie, Brian W; Buckley, David B; Smith, Brian D; Leatherman, Sarah; Paris, Brandy; Parkinson, Oliver; Parkinson, Andrew

    2015-11-01

    In the present study, we conducted a retrospective analysis of 343 in vitro experiments to ascertain whether observed (experimentally determined) values of Ki for reversible cytochrome P450 (P450) inhibition could be reliably predicted by dividing the corresponding IC₅₀ values by two, based on the relationship (for competitive inhibition) in which Ki = IC₅₀/2 when [S] (substrate concentration) = Km (Michaelis-Menten constant). Values of Ki and IC₅₀ were determined under the following conditions: 1) the concentration of P450 marker substrate, [S], was equal to Km (for IC₅₀ determinations) and spanned Km (for Ki determinations); 2) the substrate incubation time was short (5 minutes) to minimize metabolism-dependent inhibition and inhibitor depletion; and 3) the concentration of human liver microsomes was low (0.1 mg/ml or less) to maximize the unbound fraction of inhibitor. Under these conditions, predicted Ki values, based on IC₅₀/2, correlated strongly with experimentally observed Ki determinations [r = 0.940; average fold error (AFE) = 1.10]. Of the 343 predicted Ki values, 316 (92%) were within a factor of 2 of the experimentally determined Ki values, and only one value fell outside a 3-fold range. In the case of noncompetitive inhibitors, Ki values predicted from IC₅₀/2 values were overestimated by a factor of nearly 2 (AFE = 1.85; n = 13), which is to be expected because, for noncompetitive inhibition, Ki = IC₅₀ (not IC₅₀/2). The results suggest that, under appropriate experimental conditions with the substrate concentration equal to Km, values of Ki for direct, reversible inhibition can be reliably estimated from values of IC₅₀/2. PMID:26354951

  4. The Reliability of Estimating Ki Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC50 Values: A Retrospective Analysis of 343 Experiments.

    PubMed

    Haupt, Lois J; Kazmi, Faraz; Ogilvie, Brian W; Buckley, David B; Smith, Brian D; Leatherman, Sarah; Paris, Brandy; Parkinson, Oliver; Parkinson, Andrew

    2015-11-01

    In the present study, we conducted a retrospective analysis of 343 in vitro experiments to ascertain whether observed (experimentally determined) values of Ki for reversible cytochrome P450 (P450) inhibition could be reliably predicted by dividing the corresponding IC₅₀ values by two, based on the relationship (for competitive inhibition) in which Ki = IC₅₀/2 when [S] (substrate concentration) = Km (Michaelis-Menten constant). Values of Ki and IC₅₀ were determined under the following conditions: 1) the concentration of P450 marker substrate, [S], was equal to Km (for IC₅₀ determinations) and spanned Km (for Ki determinations); 2) the substrate incubation time was short (5 minutes) to minimize metabolism-dependent inhibition and inhibitor depletion; and 3) the concentration of human liver microsomes was low (0.1 mg/ml or less) to maximize the unbound fraction of inhibitor. Under these conditions, predicted Ki values, based on IC₅₀/2, correlated strongly with experimentally observed Ki determinations [r = 0.940; average fold error (AFE) = 1.10]. Of the 343 predicted Ki values, 316 (92%) were within a factor of 2 of the experimentally determined Ki values, and only one value fell outside a 3-fold range. In the case of noncompetitive inhibitors, Ki values predicted from IC₅₀/2 values were overestimated by a factor of nearly 2 (AFE = 1.85; n = 13), which is to be expected because, for noncompetitive inhibition, Ki = IC₅₀ (not IC₅₀/2). The results suggest that, under appropriate experimental conditions with the substrate concentration equal to Km, values of Ki for direct, reversible inhibition can be reliably estimated from values of IC₅₀/2.

  5. New insights into the prognostic value of Ki-67 labeling index in patients with triple-negative breast cancer.

    PubMed

    Hao, Shuang; He, Zhi-Xian; Yu, Ke-Da; Yang, Wen-Tao; Shao, Zhi-Min

    2016-04-26

    The clinicopathological importance of the Ki-67 labeling index (LI) in breast cancer has been studied intensely; however, its prognostic significance in triple-negative breast cancer (TNBC) is unclear. We aimed to determine the optimal Ki-67 cut-off point to demonstrate its prognostic relevance for breast-cancer-specific survival (BCSS) in TNBC patients. A total of 571 female TNBC patients underwent diagnosis and surgery at our institution from January 2002 to June 2011. Clinicopathological information for all patients was available and categorized by Ki-67 LI and age at diagnosis. The cut-off values for Ki-67 LI and age were selected using the medians. A varying-coefficient Cox model was used to describe the effect of Ki-67 LI on BCSS outcomes changing with age after adjustment for disease characteristics. For survival analysis, the Kaplan-Meier method and the log-rank test were used. Cox proportional hazards models were applied to determine the association of Ki-67 LI and age with BCSS outcomes after adjustment for disease characteristics. Median age was 50 years, and median Ki-67 LI was 35% (range, 0 - 97.5%). There was no prognostic significance of stratification by Ki-67 LI in all patients. When analyzing age at diagnosis as a continuous variable, the log-transformed HRKi67 > 35% vs. ≤ 35% for BCSS increased in an S-shaped curve with increasing age up to about 50 years-old and remained higher-risk for high Ki-67 LI. After adjusting for clinicopathological risk factors, low Ki-67 LI was a poor prognostic factor for BCSS (HR: 0.36, 95% CI: 0.14-0.96, P = 0.042) in patients of ≤ 50 years, but not in patients diagnosed at > 50 years (hazard ratio [HR]: 1.57, 95% CI: 0.76-3.22, P = 0.241). In conclusion, lower Ki-67 LI has poor prognosis relevance in TNBC patients diagnosed at ≤ 50 years-old. Further validation of the clinical significance of Ki-67 LI in TNBC is required. PMID:27050075

  6. New insights into the prognostic value of Ki-67 labeling index in patients with triple-negative breast cancer

    PubMed Central

    Yu, Ke-Da; Yang, Wen-Tao; Shao, Zhi-Min

    2016-01-01

    The clinicopathological importance of the Ki-67 labeling index (LI) in breast cancer has been studied intensely; however, its prognostic significance in triple-negative breast cancer (TNBC) is unclear. We aimed to determine the optimal Ki-67 cut-off point to demonstrate its prognostic relevance for breast-cancer-specific survival (BCSS) in TNBC patients. A total of 571 female TNBC patients underwent diagnosis and surgery at our institution from January 2002 to June 2011. Clinicopathological information for all patients was available and categorized by Ki-67 LI and age at diagnosis. The cut-off values for Ki-67 LI and age were selected using the medians. A varying-coefficient Cox model was used to describe the effect of Ki-67 LI on BCSS outcomes changing with age after adjustment for disease characteristics. For survival analysis, the Kaplan–Meier method and the log-rank test were used. Cox proportional hazards models were applied to determine the association of Ki-67 LI and age with BCSS outcomes after adjustment for disease characteristics. Median age was 50 years, and median Ki-67 LI was 35% (range, 0 – 97.5%). There was no prognostic significance of stratification by Ki-67 LI in all patients. When analyzing age at diagnosis as a continuous variable, the log-transformed HRKi67 > 35% vs. ≤ 35% for BCSS increased in an S-shaped curve with increasing age up to about 50 years-old and remained higher-risk for high Ki-67 LI. After adjusting for clinicopathological risk factors, low Ki-67 LI was a poor prognostic factor for BCSS (HR: 0.36, 95% CI: 0.14–0.96, P = 0.042) in patients of ≤ 50 years, but not in patients diagnosed at > 50 years (hazard ratio [HR]: 1.57, 95% CI: 0.76–3.22, P = 0.241). In conclusion, lower Ki-67 LI has poor prognosis relevance in TNBC patients diagnosed at ≤ 50 years-old. Further validation of the clinical significance of Ki-67 LI in TNBC is required. PMID:27050075

  7. Prognostic and predictive value of Ki-67 in triple-negative breast cancer

    PubMed Central

    Zhang, Peifeng; Fei, Xiaochun; Zong, Yu; Chen, Xiaosong; Huang, Ou; He, Jian-Rong; Chen, Weiguo; Li, Yafen; Shen, Kunwei; Zhu, Li

    2016-01-01

    This study was to investigate the prognostic role of Ki-67 in further classification of triple negative breast cancer (TNBC), and to test whether high expression level of Ki67 can predict benefit from carboplatin. From January 2004 to December 2012, 363 patients operated for TNBC were identified through the institutional clinical database. After a median follow-up time of 34 months (5.2–120.0 months), 62 patients (17.1%) had relapses and 33 patients (9.1%) died of breast cancer. In univariate analysis, high Ki-67 index as well as larger tumor size and lymph node involvement was associated with shorter disease-free survival (DFS) and overall survival (OS). In multivariate analysis, high Ki-67 is an independent risk factor for DFS (Risk Ratio, RR: 2.835, 95% confidence interval, 95% CI: 1.586–5.068, P < 0.001) and OS (RR: 3.180, 95% CI: 1.488–6.793, P = 0.003). When analyzing the 3-year DFS by Ki-67 distribution, Subpopulation Treatment Effect Pattern Plot analysis showed a beneficial effect of carboplatin in patients with high Ki-67 index. In conclusion, TNBC is probably a heterogeneous disease with different characteristics and prognosis, and may be further subdivided according to the Ki-67 expression levels. Patients in the high Ki- 67 group seem to benefit more from treatment with carboplatin, but this needs to be further verified. PMID:27145269

  8. Diagnostic and prognostic value of cellular proliferation assessment with Ki-67 protein in dogs suffering from benign and malignant perianal tumors.

    PubMed

    Brodzki, Adam; Łopuszyński, Wojciech; Brodzki, Piotr; Tatara, Marcin R

    2014-01-01

    In the perianal region of carnivores, skin consists of modified sebaceous glands called perianal glands. Tumors originating from perianal glands are the third most frequent type of neoplasm in male dogs after neoplastic diseases of testes and skin. Ki-67 is a nuclear non-histone protein considered a proliferation marker in normal and neoplastic proliferating cells. Previous investigations revealed that Ki-67 expression may be used as a prognostic factor for breast cancer in humans. Thus, the aim of this study was to estimate the diagnostic and prognostic value of Ki-67 evaluation in dogs suffering from benign and malignant perianal tumors. The highest value of the Ki-67 index was obtained in the carcinoma group (18.50% ± 2.68), significantly higher compared to the values obtained in the control tissue (7.63% ± 2.12) and adenoma (7.33% ± 1.06; all P < 0.05). Statistically significant differences in the Ki-67 index were not found between the epithelioma group (11.95% ± 1.96) and all other groups (P < 0.05). This investigation on dogs with perianal gland tumors has shown significantly increased expression of Ki-67 antigen in carcinoma cells, while the expression of this protein was similar in the case of control tissues, adenoma and epithelioma. Thus, it may be postulated that Ki-67 evaluation in perianal gland tumors in dogs may serve as a useful marker possessing high diagnostic and prognostic value and enabling differentiation of malignant and benign tumors. PMID:25412511

  9. Clinicopathological and Prognostic Value of Ki-67 Expression in Bladder Cancer: A Systematic Review and Meta-Analysis

    PubMed Central

    Li, Mingguo; Wu, Zhiping; Hong, Mei; Wang, Hanzhang; Svatek, Robert; Rodriguez, Ronald; Wang, Zhiping

    2016-01-01

    Background Ki-67 is an established marker of cell proliferation, and the Ki-67 index correlates with the clinical course of several cancer types, including bladder cancer (BC). However, the clinicopathological and prognostic significance of Ki-67 in bladder cancer remains unclear. Therefore, we performed a systematic review and meta-analysis to clarify this relationship. Methods A comprehensive literature search for relevant studies published up to February 1, 2016, was performed using PubMed, Cochrane Library, Embase and ISI Web of Knowledge. The effects of Ki-67 expression on survival outcome in patients with BC and BC subtypes were evaluated. Furthermore, the relationship between Ki-67 expression and the clinicopathological features of BC were assessed. Results Thirty-one studies with 5147 bladder cancer patients were selected for evaluation. Ki-67 expression was significantly associated with shorter recurrence-free (HR 1.69, 95% CI: 1.33–2.14), progression-free (HR 1.89, 95% CI: 1.43–2.51), overall (HR 2.03, 95% CI: 1.31–3.16), and cancer-specific (HR 1.69, 95% CI: 1.47–1.95) survival. Moreover, whereas high expression was more common in high tumor stage, recurrence status, tumor size, there was no correlation between high Ki-67 expression and age, gender, smoking habits, and tumor number. Importantly, analysis of the different subgroups of BC suggested that significant correlations between high Ki-67 expression and survival outcome (recurrence-free/progression-free/overall/cancer-specific survival) are present only in European-American patients. Conclusion The present results indicate that over-expression of Ki-67 is distinctly correlated with poor patient survival. Ki-67 may serve as a valuable biomarker for prognosis in BC patients, particularly in non-Asian BC patients. The results suggest no significant association between Ki-67 expression and BC prognosis in Asian patients. Further efforts are needed to fully clarify this relationship. PMID

  10. Food and value motivation: Linking consumer affinities to different types of food products.

    PubMed

    de Boer, Joop; Schösler, Hanna

    2016-08-01

    This study uses the consumer affinity concept to examine the multiple motives that may shape consumers' relationships with food. The concept was applied in a study on four broad product types in the Netherlands, which cover a wide range of the market and may each appeal to consumers with different affinities towards foods. These product types may be denoted as 'conventional', 'efficient', 'gourmet' and 'pure'. A comparative analysis, based on Higgins' Regulatory Focus Theory, was performed to examine whether food-related value motivations could explain different consumer affinities for these product types. The affinities of consumers were measured by means of a non-verbal, visual presentation of four samples of food products in a nationwide survey (n = 742) among consumers who were all involved in food purchasing and/or cooking. The affinities found could be predicted fairly well from a number of self-descriptions relating to food and eating, which expressed different combinations of type of value motivation and involvement with food. The analysis demonstrated the contrasting role of high and low involvement as well as the potential complementarity of promotion- and prevention-focused value motivation. It is suggested that knowledge of the relationships between product types, consumer affinities and value motivation can help improve the effectiveness of interventions that seek to promote healthy and sustainable diets in developed countries. PMID:27046434

  11. Food and value motivation: Linking consumer affinities to different types of food products.

    PubMed

    de Boer, Joop; Schösler, Hanna

    2016-08-01

    This study uses the consumer affinity concept to examine the multiple motives that may shape consumers' relationships with food. The concept was applied in a study on four broad product types in the Netherlands, which cover a wide range of the market and may each appeal to consumers with different affinities towards foods. These product types may be denoted as 'conventional', 'efficient', 'gourmet' and 'pure'. A comparative analysis, based on Higgins' Regulatory Focus Theory, was performed to examine whether food-related value motivations could explain different consumer affinities for these product types. The affinities of consumers were measured by means of a non-verbal, visual presentation of four samples of food products in a nationwide survey (n = 742) among consumers who were all involved in food purchasing and/or cooking. The affinities found could be predicted fairly well from a number of self-descriptions relating to food and eating, which expressed different combinations of type of value motivation and involvement with food. The analysis demonstrated the contrasting role of high and low involvement as well as the potential complementarity of promotion- and prevention-focused value motivation. It is suggested that knowledge of the relationships between product types, consumer affinities and value motivation can help improve the effectiveness of interventions that seek to promote healthy and sustainable diets in developed countries.

  12. Prognostic value of Ki67 and p53 in patients with estrogen receptor-positive and human epidermal growth factor receptor 2-negative breast cancer: Validation of the cut-off value of the Ki67 labeling index as a predictive factor

    PubMed Central

    OHARA, MASAHIRO; MATSUURA, KAZUO; AKIMOTO, ETSUSHI; NOMA, MIDORI; DOI, MIHOKO; NISHIZAKA, TAKASHI; KAGAWA, NAOKI; ITAMOTO, TOSHIYUKI

    2016-01-01

    The aim of this study was to evaluate the significance of the Ki67 labeling index and p53 status as prognostic and predictive indicators of operable estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Among 697 consecutive patients with primary breast cancer who underwent curative surgery between 2002 and 2013, 308 patients with ER-positive and HER2-negative breast cancer were assessed. The results of the multivariate Cox analysis demonstrated that a high Ki67 labeling index was significantly associated with a short recurrence-free interval (RFI) (p=0.004) and was marginally associated with a worse overall survival (p=0.074). A positive p53 status was not associated with worse outcomes. To validate the cut-off values of the Ki67 labeling index for identifying patients who may benefit from additional chemotherapy, prognostic factors were investigated in breast cancer patients treated postoperatively with endocrine therapy alone. Analysis of receiver operating characteristic curves demonstrated that a Ki67 labeling index cut-off of 20.0% was optimal for predicting recurrence among patients who did not receive adjuvant chemotherapy. The 5-year RFIs for patients with Ki67 <20 and ≥20% were 97.2 and 86.6%, respectively (p=0.0244). A high Ki67 labeling index (≥20%) was significantly associated with large tumors (p<0.01), lymph node metastasis (p=0.0236) and positive p53 status (p<0.001). The univariate analysis demonstrated that Ki67 labeling index ≥20%, lymph node metastasis and progesterone receptor negativity were significant worse prognostic factors for RFI (p=0.0333, 0.0116 and 0.0573, respectively). The Ki67 labeling index was found to be a useful prognostic factor in patients with ER-positive and HER2-negative breast cancer and the cut-off values of the Ki67 labeling index for making a decision regarding adjuvant treatment were validated. PMID:27073684

  13. Prognostic and Predictive Value of Centrally Reviewed Ki-67 Labeling Index in Postmenopausal Women With Endocrine-Responsive Breast Cancer: Results From Breast International Group Trial 1-98 Comparing Adjuvant Tamoxifen With Letrozole

    PubMed Central

    Viale, Giuseppe; Giobbie-Hurder, Anita; Regan, Meredith M.; Coates, Alan S.; Mastropasqua, Mauro G.; Dell'Orto, Patrizia; Maiorano, Eugenio; MacGrogan, Gaëtan; Braye, Stephen G.; Öhlschlegel, Christian; Neven, Patrick; Orosz, Zsolt; Olszewski, Wojciech P.; Knox, Fiona; Thürlimann, Beat; Price, Karen N.; Castiglione-Gertsch, Monica; Gelber, Richard D.; Gusterson, Barry A.; Goldhirsch, Aron

    2008-01-01

    Purpose To evaluate the prognostic and predictive value of Ki-67 labeling index (LI) in a trial comparing letrozole (Let) with tamoxifen (Tam) as adjuvant therapy in postmenopausal women with early breast cancer. Patients and Methods Breast International Group (BIG) trial 1-98 randomly assigned 8,010 patients to four treatment arms comparing Let and Tam with sequences of each agent. Of 4,922 patients randomly assigned to receive 5 years of monotherapy with either agent, 2,685 had primary tumor material available for central pathology assessment of Ki-67 LI by immunohistochemistry and had tumors confirmed to express estrogen receptors after central review. The prognostic and predictive value of centrally measured Ki-67 LI on disease-free survival (DFS) were assessed among these patients using proportional hazards modeling, with Ki-67 LI values dichotomized at the median value of 11%. Results Higher values of Ki-67 LI were associated with adverse prognostic factors and with worse DFS (hazard ratio [HR; high:low] = 1.8; 95% CI, 1.4 to 2.3). The magnitude of the treatment benefit for Let versus Tam was greater among patients with high tumor Ki-67 LI (HR [Let:Tam] = 0.53; 95% CI, 0.39 to 0.72) than among patients with low tumor Ki-67 LI (HR [Let:Tam] = 0.81; 95% CI, 0.57 to 1.15; interaction P = .09). Conclusion Ki-67 LI is confirmed as a prognostic factor in this study. High Ki-67 LI levels may identify a patient group that particularly benefits from initial Let adjuvant therapy. PMID:18981464

  14. The Value of 18F-FDG PET/CT Imaging Combined With Pretherapeutic Ki67 for Early Prediction of Pathologic Response After Neoadjuvant Chemotherapy in Locally Advanced Breast Cancer.

    PubMed

    Luo, Jurui; Zhou, Zhirui; Yang, Zhaozhi; Chen, Xingxing; Cheng, Jinyi; Shao, Zhimin; Guo, Xiaomao; Tuan, Jeffrey; Fu, Xiaolong; Yu, Xiaoli

    2016-02-01

    To evaluate the value of F-fluorodeoxyglucose-positron emission tomography/computed tomography (F-FDG PET/CT) and pretherapeutic Ki67 in predicting pathologic response in locally advanced breast cancer (LABC) after neoadjuvant chemotherapy (NAC).As a training set, total 301 LABC patients treated with NAC were retrospectively analyzed to evaluate the potential predictive value of pretherapeutic Ki67 for pathologic complete response (pCR) after NAC. Another 60 LABC patients were prospectively included as a validation set to evaluate the value of Ki67 combined PET/CT as pCR predictors. Ki67 was assessed in pretherapy core needle biopsy specimens and PET/CT scans were performed at baseline (before initiating NAC), after the 2nd, and 4th cycle of NAC. Maximum standardized uptake value (SUVmax) and its changes relative to baseline (ΔSUVmax%) were used as parameters of PEC/CT.In the training set, Ki67 was a predictor of pCR to NAC, with area under the curve (AUC) of 0.624 (P = 0.003) in receiver-operating characteristic (ROC) analysis. In the validation set, Ki67 alone did not show significant value in predicting pCR in the validation set. ΔSUVmax% after then 2nd or 4th course are predictors of pCR to NAC with the AUC of 0.774 (P = 0.002) and 0.791 (P = 0.002), respectively. When combined with ΔSUVmax% after the 2nd and 4th course NAC, Ki67 increased the value of ΔSUVmax% in predicting pCR with the AUC of 0.824 (P = 0.001). Baseline SUVmax and after 2nd, 4th course NAC had no predictive value for pCR, but SUVmax after the 2nd and 4th course showed remarkable predictive value for nonpathologic response (Grade 1 in Miller-Payne Grading System) with the AUC of 0.898 (P = 0.0001) and 0.801 (P = 0.003).Both PET/CT and Ki67 can predict pCR to NAC in LABC patients in the early phases of treatment. PET/CT combined Ki67 is a better pCR predictor for response to NAC. This helps the physician to predict the probability of pCR, and facilitates the

  15. Claudins and Ki-67

    PubMed Central

    Törzsök, Péter; Riesz, Péter; Kenessey, István; Székely, Eszter; Somorácz, Áron; Nyirády, Péter; Romics, Imre; Schaff, Zsuzsa; Lotz, Gábor

    2011-01-01

    Updated classification of urothelial cell cancer differentiates low-grade and high-grade cancers, which determines potential clinical outcome. Substantial interobserver variability necessitates new biomarkers to ensure classification. Claudins’ specific expression pattern characterizes normal tissues, different tumor types, and defined grades of tumor differentiation. The aim of this study was to examine the expression pattern of claudins and proliferation marker Ki-67 in low-grade and high-grade urothelial cell cancers compared with independent control samples of non-tumorous urothelium, as well as to reveal the predictive usefulness of claudins. The expression of claudins-1, -2, -3, -4, -5, -7, and -10 and Ki-67 was studied with quantitative immunohistochemistry and real-time RT-PCR with relative quantification in 103 samples: 86 urothelial cell cancers (27 low grade, 59 high grade) and 17 non-tumorous urothelia. Results were analyzed regarding overall survival and recurrence-free period as well. High-grade tumors overall showed significantly higher claudin-4 and Ki-67 and significantly lower claudin-7 expression when compared with low-grade ones. High-grade tumors revealed significantly shorter overall survival in Kaplan-Meier analysis. Claudin-4, claudin-7, and Ki-67 might be used as potential markers to differentiate low-grade and high-grade urothelial cell cancers, thereby possibly enhancing accuracy of pathological diagnosis and adding further information to clinical outcome. PMID:22043024

  16. Bethe Ansatz and the Spectral Theory of Affine Lie algebra-Valued Connections II: The Non Simply-Laced Case

    NASA Astrophysics Data System (ADS)

    Masoero, Davide; Raimondo, Andrea; Valeri, Daniele

    2016-09-01

    We assess the ODE/IM correspondence for the quantum g -KdV model, for a non-simply laced Lie algebra g. This is done by studying a meromorphic connection with values in the Langlands dual algebra of the affine Lie algebra g^{(1)} , and constructing the relevant {Ψ} -system among subdominant solutions. We then use the {Ψ} -system to prove that the generalized spectral determinants satisfy the Bethe Ansatz equations of the quantum g -KdV model. We also consider generalized Airy functions for twisted Kac-Moody algebras and we construct new explicit solutions to the Bethe Ansatz equations. The paper is a continuation of our previous work on the ODE/IM correspondence for simply-laced Lie algebras.

  17. Proliferative activity in human breast cancer: Ki-67 automated evaluation and the influence of different Ki-67 equivalent antibodies

    PubMed Central

    2011-01-01

    Background Ki67 labeling index (Ki67 LI), the percentage Ki67 immunoreactive cells, is a measure of tumor proliferation, with important clinical relevance in breast cancer, and it is extremely important to standardize its evaluation. Aim To test the efficacy of computer assisted image analysis (CAIA) applied to completely digitized slides and to assess its feasibility in routine practice and compare the results obtained using two different Ki67 monoclonal antibodies. Materials and methods 315 consecutive breast cancer routinely immunostained for Ki-67 (223 with SP6 and 92 with MM1 antibodies previously examined by an experienced pathologist, have been re-evaluated using Aperio Scanscope Xs. Results Mean human Ki67 LI values were 36%± 14.% and 28% ± 18% respectively for SP6 and MM1 antibodies; mean CAM Ki67 LI values were 31%± 19% and 22% ± 18% respectively for SP6 and MM1. Human and CAIA evaluation are statistically highly correlated (Pearson: 0.859, p<0.0001), although human LI are systematically higher. An interobserver variation study on CAIA performed on 84 cases showed that the correlation between the two evaluations was linear to an excellent degree. Discussion Our study shows that a) CAIA can be easily adopted in routine practice, b) human and CAIA Ki67 LI are highly correlated, although human LI are systematically higher, c) Ki67 LI using different evaluation methods and different antibodies shows important differences in cut-off values. PMID:21489202

  18. Affinity of neuroleptics for D1 receptor of human brain striatum.

    PubMed Central

    Kanba, S; Suzuki, E; Nomura, S; Nakaki, T; Yagi, G; Asai, M; Richelson, E

    1994-01-01

    We determined the inhibition-dissociation constant (Ki) of a number of neuroleptics for D1 receptors of normal human brain tissue using [3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3[benzazepine-7- ol]. SCH23390 had the highest affinity with a Ki of 0.76 nM. Among clinically used drugs, propericiazine showed the highest affinity with a Ki of 10 nM. When neuroleptics were classified according to chemical structures, the Ki values were as follows. Phenothiazines ranged from 10 nM to 250 nM. Butyrophenones ranged from 45 nM to 250 nM. Thioxanthenes ranged from 12 nM to 340 nM. Orthopramines were more than 10,000 nM. The Ki values for the binding site of this study were significantly correlated with those reported in studies using animal brain. The possible relationship between D1 receptors and negative symptoms is discussed. PMID:7918347

  19. The oxygen affinity of cytochrome bo' in Escherichia coli determined by the deoxygenation of oxyleghemoglobin and oxymyoglobin: Km values for oxygen are in the submicromolar range.

    PubMed Central

    D'Mello, R; Hill, S; Poole, R K

    1995-01-01

    Apparent oxygen affinities for Escherichia coli cells and membranes containing a terminal oxidase with only one type of ligand-binding heme, cytochrome o', were measured with oxyleghemoglobin and oxymyoglobin as sensitive oxygen reporters. Two Km values (0.15 to 0.35 microM and 0.016 to 0.085 microM) were detected, well below values determined for the purified oxidase by insensitive electrode methods. PMID:7836332

  20. Ether modifications to 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503): effects on binding affinity and selectivity for sigma receptors and monoamine transporters.

    PubMed

    Xu, Rong; Lord, Sarah A; Peterson, Ryan M; Fergason-Cantrell, Emily A; Lever, John R; Lever, Susan Z

    2015-01-01

    Two series of novel ether analogs of the sigma (σ) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the 4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moieties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for σ1 and σ2 receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT) affinity. The highest σ1 receptor affinities, Ki values of 1.75-4.63 nM, were observed for 4-O-des-methyl SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased, σ1 receptor affinity decreased, but only to a point. Allyl, propyl and bromoethyl substitutions gave σ1 receptor Ki values in the 20-30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ1 affinities, with Ki values in the 13-21 nM range. Most ligands studied exhibited comparable σ1 and σ2 affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the methylenedioxy congener showed modest six- to fourteen-fold selectivity for σ1 sites. DAT and SERT interactions proved much more sensitive than σ receptor interactions to these structural modifications. For example, the benzyl congener (σ1Ki=20.8 nM; σ2Ki=16.4 nM) showed over 100-fold higher DAT affinity (Ki=121 nM) and 6-fold higher SERT affinity (Ki=128nM) than the parent SA4503 (DAT Ki=12650 nM; SERT Ki=760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunctional ligands having a broader spectrum of possible pharmacological actions.

  1. Frequently Asked Questions on Potassium Iodide (KI)

    MedlinePlus

    ... needs to take potassium iodide (KI) after a nuclear radiation release? What potassium iodide (KI) products are currently ... needs to take potassium iodide (KI) after a nuclear radiation release? The FDA guidance prioritizes groups based on ...

  2. Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

    PubMed

    Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

    2016-03-14

    The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds.

  3. Univalent and Bivalent Ligands of Butorphan: Characteristics of the Linking Chain determine the Affinity and Potency of Such Opioid Ligands†

    PubMed Central

    Decker, Michael; Fulton, Brian S.; Zhang, Bin; Knapp, Brian I.; Bidlack, Jean M.; Neumeyer, John L.

    2009-01-01

    Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at the μ, δ, and κ opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically labile ester linkage increased stability while only partially affecting binding affinity. The resulting bivalent ligands with optimized spacer-length and structure show potent binding profiles with the most potent compound (4b) having Ki values of 0.47 nM for both the μ and κ opioid receptors, and 4a having Ki values of 0.95 and 0.62 nM for the μ and κ receptors, respectively. Both 4a and 4b were partial agonists at the κ and μ receptors in the [35S]GTPγS binding assay. PMID:19634902

  4. Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

    PubMed

    Núñez-Montenegro, Ara; Carballo, Rosa; Vázquez-López, Ezequiel M

    2014-11-01

    The binding affinities towards estrogen receptors (ERs) α and β of a set of thiosemicarbazone ligands (HL(n)) and their rhenium(I) carbonyl complexes [ReX(HL(n))(CO)3] (X=Cl, Br) were determined by a competitive standard radiometric assay with [(3)H]-estradiol. The ability of the coordinated thiosemicarbazone ligands to undergo deprotonation and the lability of the ReX bond were used as a synthetic strategy to obtain [Re(hpy)(L(n))(CO)3] (hpy=3- or 4-hydroxypyridine). The inclusion of the additional hpy ligand endows the new thiosemicarbazonate complexes with an improved affinity towards the estrogen receptors and, consequently, the values of the inhibition constant (Ki) could be determined for some of them. In general, the values of Ki for both ER subtypes suggest an appreciable selectivity towards ERα.

  5. Binding affinity and agonist activity of putative endogenous cannabinoids at the human neocortical CB1 receptor.

    PubMed

    Steffens, Marc; Zentner, Josef; Honegger, Jürgen; Feuerstein, Thomas J

    2005-01-01

    We investigated the affinity of putative endocannabinoids (2-arachidonylglycerol, 2-AG; noladin ether, virodhamine) for the human neocortical CB1 receptor. Functional activity of these compounds (including anandamide, AEA) was determined by examining basal and forskolin-stimulated cAMP formation. Assays were performed with synaptosomes, prepared from fresh human neocortical tissue. Receptor affinity was assessed from competition binding experiments with the CB1/2 agonist [3H]-CP55.940 in absence or presence of a protease inhibitor to assess enzymatic stability. Noladin ether and virodhamine inhibited [3H]-CP55.940 binding (Ki: 98, 1740 nM, respectively). Protease inhibition decreased the Ki value of virodhamine (Ki: 912 nM), but left that of noladin ether unchanged. 2-AG almost lacked affinity (Ki lymphoblasic )10 microM). Basal cAMP formation was unaffected by AEA and noladin ether, but strongly enhanced by 2-AG and virodhamine. Forskolin-stimulated cAMP formation was inhibited by AEA and noladin ether (IC50: 69, 427 nM, respectively) to the same extent as by CP55.940 (Imax each approximately 30%). Inhibitions by AEA or noladin ether were blocked by the CB1 receptor antagonist AM251. Virodhamine increased forskolin-stimulated cAMP formation, also in presence of AM251, by approximately 20%. 2-AG had no effect; in presence of AM251, however, 10 microM 2-AG stimulated cAMP formation by approximately 15%. Our results suggest, that AEA and noladin ether are full CB1 receptor agonists in human neocortex, whereas virodhamine may act as a CB1 receptor antagonist/inverse agonist. Particularly the (patho)physiological role of 2-AG should be further investigated, since its CB1 receptor affinity and agonist activity especially in humans might be lower than generally assumed. PMID:15588725

  6. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  7. Bethe Ansatz and the Spectral Theory of Affine Lie Algebra-Valued Connections I. The simply-laced Case

    NASA Astrophysics Data System (ADS)

    Masoero, Davide; Raimondo, Andrea; Valeri, Daniele

    2016-06-01

    We study the ODE/IM correspondence for ODE associated to {widehat{mathfrak{g}}}-valued connections, for a simply-laced Lie algebra {mathfrak{g}}. We prove that subdominant solutions to the ODE defined in different fundamental representations satisfy a set of quadratic equations called {Ψ}-system. This allows us to show that the generalized spectral determinants satisfy the Bethe Ansatz equations.

  8. Immunolocalization of Ki-67 in different periodontal conditions

    PubMed Central

    Preethi, Penubolu Lakshmi; Rao, Suresh Rango; Madapusi, Balaji Thodur; Narasimhan, Malathi

    2014-01-01

    Background: Ki-67 which is a non-histone nuclear protein which is expressed in proliferating cells, during all the active phases of the cell cycle. Increased Ki-67 expression has been seen in several inflammatory and malignant conditions like diabetes, rheumatoid arthritis, atherosclerosis, pancreatitis and squamous cell carcinoma. Aim: The aim of the present study is to analyze the expression of Ki-67 in gingival tissues by immunohistochemistry in smokers and non-smokers with healthy gingiva and chronic periodontitis. Materials and Methods: Gingival biopsies (n = 32) were obtained from smokers who had clinically healthy gingiva (n = 8), smokers with periodontitis (n = 8), chronic periodontitis (n = 8) and healthy gingiva (n = 8). The expression of Ki-67 was evaluated immunohistochemically. Statistical analysis used: Mean and standard deviation were estimated for the gingival tissue extract sample for each study group. Mean values were compared between different study groups by, one way ANOVA, post hoc analysis. In this study P < 0.05 was considered as the level of significance. Results: The mean number of Ki-67 positive cells/field was higher in the smokers with periodontitis group. When the mean Ki-67 positive cells were compared between different groups, statistical significant difference was observed between healthy and both the periodontitis groups (P = 0.000) and between smokers group (P = 0.001). Conclusions: Ki-67 was maximally expressed in smoker with periodontitis followed by chronic periodontitis patients, healthy smokers and healthy control patients which shed light on the toxic effects of tobacco in dysregulating the cell cycle and cellular proliferation. The findings of this study also help us to understand the role of the cell cycle in resolution of periodontal inflammation which is a salient feature in the pathogenesis of chronic periodontitis. PMID:24872622

  9. KW-3902, a selective high affinity antagonist for adenosine A1 receptors.

    PubMed Central

    Nonaka, H.; Ichimura, M.; Takeda, M.; Kanda, T.; Shimada, J.; Suzuki, F.; Kase, H.

    1996-01-01

    1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively. PMID:8732272

  10. Ki-67 expression in axillary lymph node metastases in breast cancer is prognostically significant.

    PubMed

    Tawfik, Kareem; Kimler, Bruce F; Davis, Marilyn K; Fan, Fang; Tawfik, Ossama

    2013-01-01

    Several studies have documented the prognostic significance of cell proliferation in breast cancer and its positive relationship with tumor grade, size, mitotic activity, hormonal and Her-2 status, and tumor progression. The Ki-67 antigen provides an accurate measure of the growth fraction of a tumor. Ki-67 expression in 103 primary breast carcinomas and their corresponding axillary lymph node metastases was correlated with age, tumor grade, size, estrogen receptor (ER), progesterone receptor (PgR), p53, epidermal growth factor receptor (EGFR), Bcl-2, Her-2 status, and patients' overall survival. Median Ki-67 expression in primary and metastatic tumors was 20% and 15%, respectively. Although there was no difference in overall survival (P = .65, log-rank test) between primary tumors with less than or at least 10% Ki-67 expression, there was significantly better overall survival when Ki-67 expression in lymph nodes was less than 10% (P = .040). For patients whose primary tumors exhibited Ki-67 expression less than 10%, most of their metastatic lesions had a similar low Ki-67; these patients had a favorable outcome. A small subgroup was noted to have a nodal Ki-67 of 10% or more and worse survival (P = .047). For patients whose primary tumors had a Ki-67 of 10% or more, most of their metastatic lesions had similar high Ki-67 values; however, a group of 12 patients had lymph node Ki-67 less than 10% and had a better overall survival (P = .092). Our results showed that measurement of Ki-67 in lymph node is superior to its evaluation in primary tumors. Identification of subgroups of patients in whom Ki-67 expression in lymph nodes differs from expression in primary tumor may assist in the selection of therapeutic options.

  11. Structures and Electronic Properties of (KI)n(-/0) (n = 1-4) and K(KI)n(-/0) (n = 1-3) Clusters: Photoelectron Spectroscopy, Isomer-Depletion, and ab Initio Calculations.

    PubMed

    Hou, Gao-Lei; Feng, Gang; Zhao, Li-Juan; Xu, Hong-Guang; Zheng, Wei-Jun

    2015-11-12

    The (KI)n(-) (n = 1-4) and K(KI)n(-) (n = 1-3) clusters were studied by negative ion photoelectron spectroscopy and ab initio calculations. Comparison between the theoretical vertical detachment energies and the experimental values revealed that multiple isomers may coexist in the experiments. The existence of two isomers for K(KI)(-) and K(KI)2(-) were confirmed directly by isomer-depletion experiments, in which the low adiabatic detachment energy isomers were depleted by a 1064 nm laser beam before the anions were photodetached by a 532 nm laser beam. Our results show that the most stable structures of the K(KI)(-), (KI)2(-), and K(KI)2(-) anions are chain structures, while those of their neutral counterparts are planar. Three-dimensional structures start to appear at n = 3 for (KI)n(-/0) and K(KI)n(-/0). In the K(KI)n(-) cluster anions, the excess electron is localized on the extra K atom and forms an electron pair with the existing s electron of the K atom; the resulting negatively charged K prefers to interact with the other positively charged K atoms rather than with the I atoms. Both the anionic and neutral (KI)4 clusters have cuboid structures, which may be regarded as the smallest structural motif of KI crystal. PMID:26473992

  12. Philosophy, psychology, physics and practice of ki.

    PubMed

    Ohnishi, S Tsuyoshi; Ohnishi, Tomoko

    2009-06-01

    Ki (in Japanese) or Qi (in Chinese) is the key concept in Eastern medicine, Eastern philosophy, as well as in martial arts. We explain the philosophical and psychological background of Ki. We emphasize that the unique aspects of Eastern philosophy are 'non-linearity' and 'holistic' approach. We then present physics aspect of Ki. Our experiments demonstrated that a 'Ki-beam' carries 'entropy' (or information), which is different from 'energy'. We introduce our experience of having taught Ki to 37 beginners in the United States through the Nishino Breathing Method. If beginners had martial arts training or a strong background in music or dance, about half of them could sense Ki within 10 weeks (1 h class per week) of practice. PMID:18955316

  13. Philosophy, Psychology, Physics and Practice of Ki

    PubMed Central

    Ohnishi, Tomoko

    2009-01-01

    Ki (in Japanese) or Qi (in Chinese) is the key concept in Eastern medicine, Eastern philosophy, as well as in martial arts. We explain the philosophical and psychological background of Ki. We emphasize that the unique aspects of Eastern philosophy are ‘non-linearity’ and ‘holistic’ approach. We then present physics aspect of Ki. Our experiments demonstrated that a ‘Ki-beam’ carries ‘entropy’ (or information), which is different from ‘energy’. We introduce our experience of having taught Ki to 37 beginners in the United States through the Nishino Breathing Method. If beginners had martial arts training or a strong background in music or dance, about half of them could sense Ki within 10 weeks (1 h class per week) of practice. PMID:18955316

  14. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  15. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

    PubMed

    Habicht, K-L; Singh, N S; Indig, F E; Wainer, I W; Moaddel, R; Shimmo, R

    2015-09-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  16. Impact of intratumoural heterogeneity on the assessment of Ki67 expression in breast cancer.

    PubMed

    Aleskandarany, M A; Green, A R; Ashankyty, I; Elmouna, A; Diez-Rodriguez, M; Nolan, C C; Ellis, I O; Rakha, E A

    2016-07-01

    In breast cancer (BC), the prognostic value of Ki67 expression is well-documented. Intratumoural heterogeneity (ITH) of Ki67 expression is amongst the several technical issues behind the lag of its inclusion into BC prognostic work-up. The immunohistochemical (IHC) expression of anti-Ki67 antibody (MIB1 clone) was assessed in four full-face (FF) sections from different primary tumour blocks and their matched axillary nodal (LN) metastases in a series of 55 BC. Assessment was made using the highest expression hot spots (HS), lowest expression (LS), and overall/average expression scores (AS) in each section. Heterogeneity score (Hes), co-efficient of variation, and correlation co-efficient were used to assess the levels of Ki67 ITH. Ki67 HS, LS, and AS scores were highly variable within the same section and between different sections of the primary tumour, with maximal variation observed in the LS (P < 0.001). The least variability between the different slides was observed with HS scoring. Although the associations between Ki67 and clinicopathological and molecular variables were similar when using HS or AS, the best correlation between AS and HS was observed in tumours with high Ki67 expression only. Ki67 expression in LN deposits was less heterogeneous than in the primary tumours and was perfectly correlated with the HS Ki67 expression in the primary tumour sections (r = 0.98, P < 0.001). In conclusion, assessment of Ki67 expression using HS scoring method on a full-face BC tissue section can represent the primary tumour growth fraction that is likely to metastasise. The association between Ki67 expression pattern in the LN metastasis and the HS in the primary tumour may reflect the temporal heterogeneity through clonal expansion. PMID:27380874

  17. Synthesis and NMDA receptor affinity of fluorinated dioxadrol analogues.

    PubMed

    Banerjee, Ashutosh; Schepmann, Dirk; Wünsch, Bernhard

    2010-06-01

    A series of dioxadrol analogues with fluorine substituents in position 4 of the piperidine ring has been synthesized and pharmacologically evaluated. The key step in the synthesis was the fluorination of diastereomeric piperidones 6a and 6c as well as diastereomeric alcohols 9a and 9c with DAST. The reaction of the alcohols 9a and 9c took place with inversion of configuration. After removal of the Cbz-protective group, the NMDA receptor affinities of the resulting secondary amines 8a, 8c, 12b, and 12d were investigated in receptor binding studies. It was shown that the like-configuration of the ring junction was crucial for high NMDA receptor affinity. An axially oriented fluorine atom in position 4 led to 2-(2,2-diphenyl-1,3-dioxolan-4-yl)-4-fluoropiperidine (12d, WMS-2517) with a K(i)-value of 27nM. The NMDA receptor affinity of 8c (WMS-2513) with an additional fluorine atom in equatorial 4-position was slightly reduced (K(i)=81 nM). Both fluorinated dioxadrol derivatives 8c and 12d showed high selectivity against sigma(1) and sigma(2) receptors as well as the polyamine binding site of NR2B receptors.

  18. Role of Ki-67 in acromegalic patients with hyperprolactinemia: retrospective analysis in 61 Chinese Patients.

    PubMed

    Huan, Cheng; Cui, Guihua; Lu, Chao; Qu, Xin; Han, Tao

    2015-03-01

    To evaluate the specific characteristics in acromegalic patients with hyperprolactinemia by analyzing the differences between patients with different Ki-67 values. Between 2002 and 2010, a set of data on 61 patients undergoing transsphenoidal surgery was available at the Department of Neurosurgery, Provincial Hospital Affiliated to Shandong University. Patients were divided into Ki-67 >3% group and <3% group. A retrospective analysis of clinical, hormonal, immunohistochemical, and imaging was observed in all patients. There were no significant differences in age, gender, tumor size and apoplexy between the two groups. Time interval in Ki-67 ≥3% group was longer than <3% group (P=0.037). Patients in Ki-67 >3% group had a higher rate of invasiveness (P=0.048), higher incidences of diabetes mellitus (P=0.036), coarse facial features (P=0.048), large hands and feet (P=0.003), higher GH levels (P<0.05), higher diabetes insipidus rate (P<0.001), and more frequent recurrence (P=0.011) than Ki-67 <3% group. Patients with higher Ki-67 value harbored longer time interval, more aggressive tumors, more acromegaly manifestations, higher GH level, and higher recurrence than patients with lower Ki-67 value. PMID:25796164

  19. KI: A tool for knowledge integration

    SciTech Connect

    Murray, K.S.

    1996-12-31

    Knowledge integration is the process of incorporating new information into a body of existing knowledge. It involves determining how new and existing knowledge interact and how existing knowledge should be modified to accommodate the new information. KI is a machine learning program that performs knowledge integration. Through actively investigating the interaction of new information with existing knowledge KI is capable of detecting and exploiting a variety of diverse learning opportunities during a single learning episode. Empirical evaluation suggests that KI provides significant assistance to knowledge engineers while integrating new information into a large knowledge base.

  20. Synthesis and Binding Affinity of Novel Mono- and Bivalent Morphinan Ligands for κ, μ and δ Opioid Receptors

    PubMed Central

    Zhang, Bin; Zhang, Tangzhi; Sromek, Anna W.; Scrimale, Thomas; Bidlack, Jean M.

    2011-01-01

    A novel series of homo- and heterodimeric ligands containing κ/μ agonist and μ agonist/antagonist pharmacophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro binding affinity at κ, μ, and δ opioid receptors and their functional activities were determined at κ and μ receptors in [35S]GTPγS functional assays. Most of these compounds had high binding affinity at μ and κ receptors (Ki values less than 1 nM). Compound 15b, which contains butorphan (1) at one end of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with binding affinity Ki values of 0.089 nM at the μ receptor and 0.073 nM at the κ receptor. All of the morphinan-derived ligands were found to be partial κ and μ agonists; ATPM-derived ligands 12 and 11 were found to be full κ agonists and partial μ agonists. PMID:21482470

  1. Affinities of pirenzepine for muscarinic cholinergic receptors in membranes isolated from bovine tracheal mucosa and smooth muscle

    SciTech Connect

    Madison, J.M.; Jones, C.A.; Tom-Moy, M.; Brown, J.K.

    1987-03-01

    Muscarinic cholinergic receptors have been classified into subtypes based on their high (M-1 subtype) or low (M-2 subtype) affinities for the nonclassic antagonist pirenzepine, and this classification has important experimental and therapeutic implications. Because muscarinic receptors are abundant in the airways where they mediate several different cellular responses, the goal of this study was to characterize the affinities of pirenzepine for the muscarinic receptors in bovine tracheal mucosa and smooth muscle. After isolating membrane particulates from mucosa and smooth muscle, as well as from bovine cerebral cortex (a known source of M-1 receptors), we used /sup 3/H-quinuclidinyl benzilate to label muscarinic receptors in the particulates and performed competition radioligand binding assays in the presence of either atropine or pirenzepine. Receptors from all 3 tissues (mucosa, smooth muscle, and cerebral cortex) were of a relatively uniform affinity for atropine (range of KI values: 0.8 +/- 0.4 X 10(-9) to 2.4 +/- 1.7 X 10(-9) M), as would be predicted for this classic muscarinic antagonist. By contrast, affinities for pirenzepine differed depending on the tissue. In cerebral cortex, the majority of receptors were of high affinity for pirenzepine (KI = 1.8 +/- 1.4 X 10(-8) M). In both mucosa and smooth muscle, receptors were of low affinity for pirenzepine (Kl = 4.8 +/- 0.4 to 6.9 +/- 3.8 X 10(-7) M). We conclude that muscarinic cholinergic receptors in bovine tracheal mucosa and smooth muscle are predominantly of the M-2 subtype.

  2. Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand.

    PubMed

    Huang, Xinyi

    2003-02-01

    For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception; in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K(i) values that can be resolved is the K(d) value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected.

  3. Automating proliferation rate estimation from Ki-67 histology images

    NASA Astrophysics Data System (ADS)

    Al-Lahham, Heba Z.; Alomari, Raja S.; Hiary, Hazem; Chaudhary, Vipin

    2012-03-01

    Breast cancer is the second cause of women death and the most diagnosed female cancer in the US. Proliferation rate estimation (PRE) is one of the prognostic indicators that guide the treatment protocols and it is clinically performed from Ki-67 histopathology images. Automating PRE substantially increases the efficiency of the pathologists. Moreover, presenting a deterministic and reproducible proliferation rate value is crucial to reduce inter-observer variability. To that end, we propose a fully automated CAD system for PRE from the Ki-67 histopathology images. This CAD system is based on a model of three steps: image pre-processing, image clustering, and nuclei segmentation and counting that are finally followed by PRE. The first step is based on customized color modification and color-space transformation. Then, image pixels are clustered by K-Means depending on the features extracted from the images derived from the first step. Finally, nuclei are segmented and counted using global thresholding, mathematical morphology and connected component analysis. Our experimental results on fifty Ki-67-stained histopathology images show a significant agreement between our CAD's automated PRE and the gold standard's one, where the latter is an average between two observers' estimates. The Paired T-Test, for the automated and manual estimates, shows ρ = 0.86, 0.45, 0.8 for the brown nuclei count, blue nuclei count, and proliferation rate, respectively. Thus, our proposed CAD system is as reliable as the pathologist estimating the proliferation rate. Yet, its estimate is reproducible.

  4. Significance of IMP3, nucleophosmin, and Ki-67 expression in papillary thyroid carcinoma.

    PubMed

    Yorukoglu, Aygun; Yalcin, Nagihan; Avci, Arzu; Cakalagaoglu, Fulya; Yaylali, Guzin; Akin, Fulya; Haciyanli, Mehmet; Ozden, Akin

    2015-02-01

    The purpose of our study was to investigate the diagnostic value of expression of IMP3, nucleophosmin, and correlation of these markers with Ki-67 proliferation index in papillary thyroid carcinoma and benign neoplasms of thyroid gland. The aim was also to investigate whether there is a difference between papillary and micropapillary carcinomas with regard to clinicopathologic parameters beside IMP3, nucleophosmin, and Ki-67 proliferation index. It was concluded that IMP3 and nucleophosmin cannot be a routine diagnostic marker for discrimination of papillary carcinomas and benign lesions. IMP3 positive staining was quite scarce in IMP3 positive papillary carcinomas although specifity of IMP3 is 100%. A statistically significant correlation was not detected between nucleophosmin, IMP-3, and Ki-67 proliferation index. A statistically significant correlation was found between tumor size, lymphovascular embolism, and Ki-67 proliferation index. There was also significant correlation between tumor size and lymphovascular embolism.

  5. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  6. Development of 3-Phenyltropane Analogs with High Affinity for the Dopamine and Serotonin Transporters and Low Affinity for the Norepinephrine Transporter

    PubMed Central

    Jin, Chunyang; Navarro, Hernán A.; Carroll, F. Ivy

    2010-01-01

    Previous studies showed that the mixed monoamine transporter inhibitor (6, RTI-112) reduced cocaine self-administration at a high level of serotonin transporter (5-HTT) occupancy with no detectable dopamine transporter (DAT) occupancy. In this study, a series of 3β-(substituted phenyl)tropane-2β-carboxylic acid methyl esters 7a-g, 3β-(4-methoxyphenyl)tropane-2β-carboxylic acid esters 8a-j, and 3β-(4-methoxyphenyl)-2β-[3-(4′-methylphenyl)isoxazol-5-yl]tropane (9) were synthesized and evaluated for their monoamine transporter binding affinities to identify potent and selective compounds for both the DAT and 5-HTT relative to the norepinephrine transporter (NET). A number of compounds showed high binding affinities for both the DAT and 5-HTT and low affinity for the NET. 3β-(4-Methoxyphenyl)tropane-2β-carboxylic acid 2-(3-iodo-4-aminophenyl)ethyl ester (8i) with an IC50 value of 2.5 nM for the DAT and Ki values of 3.5 nM and 2040 nM for the 5-HTT and NET, respectively, is the most potent and selective compound for the DAT and 5-HTT relative to the NET in this study. PMID:19053748

  7. Novel high-affinity and selective biaromatic 4-substituted gamma-hydroxybutyric acid (GHB) analogues as GHB ligands: design, synthesis, and binding studies.

    PubMed

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte; Frydenvang, Karla; Dahl, Ivar F; Bräuner-Osborne, Hans; Brehm, Lotte; Frølund, Bente; Clausen, Rasmus P

    2008-12-25

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites in brain, of which the latter have not been linked unequivocally to function, but are speculated to be GHB receptors. In this study, a series of biaromatic 4-substituted GHB analogues, including 4'-phenethylphenyl, 4'-styrylphenyl, and 4'-benzyloxyphenyl GHB analogues, were synthesized and characterized pharmacologically in a [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([3H]NCS-382) binding assay and in GABA(A) and GABA(B) receptor binding assays. The compounds were selective for the high-affinity GHB binding sites and several displayed Ki values below 100 nM. The affinity of the 4-[4'-(2-iodobenzyloxy)phenyl] GHB analogue 17b was shown to reside predominantly with the R-enantiomer (Ki = 22 nM), which has higher affinity than previously reported GHB ligands.

  8. One-step purification of lactoperoxidase from bovine milk by affinity chromatography.

    PubMed

    Atasever, Ali; Ozdemir, Hasan; Gulcin, Ilhami; Irfan Kufrevioglu, O

    2013-01-15

    Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC(50) of 0.848.10(-5)M. The K(i) for sulphanilamide was determined to be 3.57.10(-5)M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R(z) (A(412)/A(280)) value. The R(z) value for the purified LPO was found to be 0.7. Maximum binding was achieved and K(m) and V(max) values were determined.

  9. Quantitative Structure-Activity Relationship for High Affinity 5-HT1A Receptor Ligands Based on Norm Indexes.

    PubMed

    Jia, Qingzhu; Cui, Xue; Li, Lei; Wang, Qiang; Liu, Ying; Xia, Shuqian; Ma, Peisheng

    2015-12-24

    Arylpiperazine derivatives are promising 5-hydroxytryptamine (5-HT) receptor ligands which can inhibit serotonin reuptake effectively. In this work, some norm index descriptors were proposed and further utilized to develop a model for predicting 5-HT1A receptor affinity (pKi) of 88 arylpiperazine derivatives. Results showed that this new model could provide satisfactory predictions with the square of the correction coefficient (R(2)) of 0.8891 and the squared correlation coefficient of cross-validation (Q(2)) of 0.8082, respectively. In addition, the applicability domain of this model was validated by using the leverage approach and results which suggested potential large scale for further utilization of this model. The results of statistical values and validation tests demonstrated that our proposed norm index based model could be successfully applied for predicting the affinity 5-HT1A receptor ligands of arylpiperazine derivatives.

  10. Transformation in Dang-ki Healing: The Embodied Self and Perceived Legitimacy.

    PubMed

    Lee, Boon-Ooi

    2016-09-01

    Since spirit possession in mediumship and shamanism resembles psychotic symptoms, early researchers perceived spirit mediums and shamans as psychiatric patients whose psychopathology was culturally sanctioned. However, other researchers have not only challenged this assumption, but also proposed that spirit possession has transformative benefits. The idiom of spirit possession provides cultural meanings for spirit mediums and shamans to express and transform their personal experiences. The present case study focuses on dang-ki healing, a form of Chinese mediumship practiced in Singapore, in which a deity possesses a human (i.e., dang-ki) to offer aid to supplicants. This study seeks to explore whether involvement in dang-ki healing is transformative; and if so, how the dang-ki's transformation is related to his self and the perceived legitimacy of his mediumship. At a shrine, I interviewed 20 participants, including a male dang-ki, 10 temple assistants, and nine clients. The results obtained were supportive of the therapeutic nature of spirit possession. First, there is a relationship between his self-transformation and the perceived legitimacy of his mediumship. As his clients and community have recognized his spirit possession as genuine, and the healing power of his possessing god, he is able to make use of mediumship as a means for spiritual development. Second, he has developed his spirituality by internalizing his god's positive traits (e.g., compassion). Deities worshipped in dang-ki healing can be conceptualized as ideal selves who represent a wide range of positive traits and moral values of Chinese culture. Thus, the possession of a deity is the embodiment of an ideal self. Finally, the dang-ki's transformation may run parallel to his god's transformation. In Chinese religions, gods have to constantly develop their spirituality even though they are already gods. An understanding of the god's spiritual development further sheds light on the dang-ki's self

  11. Transformation in Dang-ki Healing: The Embodied Self and Perceived Legitimacy.

    PubMed

    Lee, Boon-Ooi

    2016-09-01

    Since spirit possession in mediumship and shamanism resembles psychotic symptoms, early researchers perceived spirit mediums and shamans as psychiatric patients whose psychopathology was culturally sanctioned. However, other researchers have not only challenged this assumption, but also proposed that spirit possession has transformative benefits. The idiom of spirit possession provides cultural meanings for spirit mediums and shamans to express and transform their personal experiences. The present case study focuses on dang-ki healing, a form of Chinese mediumship practiced in Singapore, in which a deity possesses a human (i.e., dang-ki) to offer aid to supplicants. This study seeks to explore whether involvement in dang-ki healing is transformative; and if so, how the dang-ki's transformation is related to his self and the perceived legitimacy of his mediumship. At a shrine, I interviewed 20 participants, including a male dang-ki, 10 temple assistants, and nine clients. The results obtained were supportive of the therapeutic nature of spirit possession. First, there is a relationship between his self-transformation and the perceived legitimacy of his mediumship. As his clients and community have recognized his spirit possession as genuine, and the healing power of his possessing god, he is able to make use of mediumship as a means for spiritual development. Second, he has developed his spirituality by internalizing his god's positive traits (e.g., compassion). Deities worshipped in dang-ki healing can be conceptualized as ideal selves who represent a wide range of positive traits and moral values of Chinese culture. Thus, the possession of a deity is the embodiment of an ideal self. Finally, the dang-ki's transformation may run parallel to his god's transformation. In Chinese religions, gods have to constantly develop their spirituality even though they are already gods. An understanding of the god's spiritual development further sheds light on the dang-ki's self-transformation.

  12. Uptake of ozone to deliquesced KI and mixed KI/NaCl aerosol particles.

    PubMed

    Rouvière, Aurélie; Sosedova, Yulia; Ammann, Markus

    2010-07-01

    The kinetics of uptake of ozone to deliquesced potassium iodide (KI) aerosol particles has been investigated in an aerosol flow tube at 72-75% relative humidity, room temperature, and atmospheric pressure. The observed loss of ozone was further analyzed in terms of a numeric model to explicitly track the iodide concentration in the particles. This allowed retrieving a value alpha(b) = 0.6 +/- (0.5)(0.4) for the bulk accommodation coefficient (alpha(b)). The second order rate constant in the bulk phase agreed with available literature (k(b) = (1.0 +/- 0.3) x 10(9) M(-1) s(-1)) even for the high ionic strength conditions of the present experiments. As long as iodide remained in excess, the average uptake coefficient was gamma = (1.10 +/- 0.20) x 10(-2). Different experiments were performed where the iodide to chloride ratio, the ozone concentration, and the surface to volume ratio of particles were varied. In combination, the results obtained indicate that uptake was driven by fast bulk accommodation and reaction in the bulk for all conditions investigated. The results further suggest that ozone uptake is not limited by the bulk accommodation coefficient alpha(b) under atmospheric conditions.

  13. The cell proliferation antigen Ki-67 organises heterochromatin

    PubMed Central

    Sobecki, Michal; Mrouj, Karim; Camasses, Alain; Parisis, Nikolaos; Nicolas, Emilien; Llères, David; Gerbe, François; Prieto, Susana; Krasinska, Liliana; David, Alexandre; Eguren, Manuel; Birling, Marie-Christine; Urbach, Serge; Hem, Sonia; Déjardin, Jérôme; Malumbres, Marcos; Jay, Philippe; Dulic, Vjekoslav; Lafontaine, Denis LJ; Feil, Robert; Fisher, Daniel

    2016-01-01

    Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. DOI: http://dx.doi.org/10.7554/eLife.13722.001 PMID:26949251

  14. Quantifying Affinity among Chinese Dialects.

    ERIC Educational Resources Information Center

    Cheng, Chin-Chuan

    A study of the relationships between Chinese dialects based on a quantitative measure of dialect affinity is summarized. First, tone values in all the dialect localities available in the early 1970s were used to calculate the dialectal differences in terms of tone height with respect to the "yin and yang" split. In the late 1970s, calculations of…

  15. Coexistence of HER2, Ki67, and p53 in Osteosarcoma: A Strong Prognostic Factor

    PubMed Central

    Mardanpour, Keykhosro; Rahbar, Mahtab; Mardanpour, Sourena

    2016-01-01

    Background: Many laboratories are currently evaluating the usefulness of the determination of human epidermal growth factor receptor 2 (HER2), p53, and Ki67 proliferation indices using immunohistochemical techniques in cancer. Although the available studies suggest that these factors might indeed be helpful in making treatment decisions in osteosarcoma patients, their clinical usefulness is still controversial. Aims: We proposed to introduce the value of the coexistence of HER2 overexpression, p53 protein accumulation, and Ki67 in osteosarcoma, which could be a prognostic factor in osteosarcoma. Material and Methods: Expression of HER2, p53, and Ki67 was examined by immunohistochemistry in samples of resected bone tumor tissue from 56 patients with osteosarcoma, obtained between 2009 and 2014 (median follow-up period of 48 months), and their significance for prognosis was analyzed. Results: Of the 56 osteogenic sarcoma tissue samples, 80, 89, and 96.5% were positive for HER2 overexpression, p53 protein accumulation, and Ki67 expression, respectively. Overexpression of HER2 and accumulation of p53 protein significantly correlated with reduced disease-free (P < 0.01) and overall survival (P < 0.003). HER2 and Ki67 co-overexpression significantly correlated with decreased disease-free (P < 0.03) and overall survival (P < 0.02). HER2, accumulation of p53 protein, and Ki67 co-overexpression significantly correlated with reduced disease-free (P < 0.01) and overall survival (P < 0.005) as did patients with larger tumor size, high grade of tumor, positive lymph node, and metastasis status within the specified period of follow up. Conclusions: We found evidence that coexistence of HER2 and Ki67 overexpression and p53 protein accumulation predict the development of lymph node involvement and metastases in patients with high-grade osteosarcoma and were significantly associated with reduced survival. PMID:27298815

  16. Design of peptoid analogue dimers and measure of their affinity for Grb2 SH3 domains.

    PubMed

    Vidal, M; Liu, W-Q; Lenoir, C; Salzmann, J; Gresh, N; Garbay, C

    2004-06-15

    This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.

  17. What is the Prognostic Significance of Ki-67 Positivity in Oral Squamous Cell Carcinoma?

    PubMed Central

    Xie, Shang; Liu, Ying; Qiao, Xue; Hua, Rui-Xi; Wang, Kan; Shan, Xiao-Feng; Cai, Zhi-Gang

    2016-01-01

    BACKGROUD: Numerous studies have stated that Ki-67 is a good prognostic marker in oral squamous cell carcinoma (OSCC). However, some researchers believe the contrary. To address this controversy, we performed a systematic literature retrieval to estimate the prognostic significance of Ki-67 expression in patients with OSCC. METHODS: Databases covering Pubmed, Ovid, Web of Science, Embase and the Cochrane library were searched regardless of publication year. Overall survival (OS), local recurrence (LR) and disease-free survival (DFS) were the main outcome measures. Relative risks (RRs) and its 95% confidential intervals (CIs) were used for statistical analysis. RESULTS: Twenty-seven articles with 2146 patients were included in this study. The results of the meta-analysis suggested that the pooled RRs and its CIs for OS, LR, and DFS were 1.45 (1.15 - 1.84), 1.76 (0.74 - 4.16) and 1.52 (1.07 - 2.14), respectively. However, the heterogeneities of OS and LR were obvious (I-squared (OS) = 59.4%, I-squared (LR) = 72.6%). After subgroup analysis based on systemic treatment, the cut-off value of Ki-67 expression, ethnicity and types of antibody, the heterogeneities became acceptable. It was observed that systemic treatment, cut-off values of Ki-67 expression, ethnicity and the types of antibody affected the results. The statistical analyses of subgroups suggested that non-systemic treatment, (OR=1.77, 95% CI = 1.39-2.25, p = 0.000) and Asian populations (OR=2.09, 95% CI = 1.32-3.32, p = 0.002) are high risks for Ki-67 high expression, and low cut-off value of Ki-67 expression (OR = 1.44, 95% CI = 1.001-2.072), MIB-1 antibody (OR = 1.48, OR 95% = 1.10-1.99) might affect the identification of results. CONCLUSIONS: According to this meta-analysis, high Ki-67 expression might be a negative prognostic marker of patients with OSCC, especially in Asian populations. In addition, Ki-67 expression affects the treatment response. PMID:27162533

  18. Bimodality of intratumor Ki67 expression is an independent prognostic factor of overall survival in patients with invasive breast carcinoma.

    PubMed

    Laurinavicius, Arvydas; Plancoulaine, Benoit; Rasmusson, Allan; Besusparis, Justinas; Augulis, Renaldas; Meskauskas, Raimundas; Herlin, Paulette; Laurinaviciene, Aida; Abdelhadi Muftah, Abir A; Miligy, Islam; Aleskandarany, Mohammed; Rakha, Emad A; Green, Andrew R; Ellis, Ian O

    2016-04-01

    Proliferative activity, assessed by Ki67 immunohistochemistry (IHC), is an established prognostic and predictive biomarker of breast cancer (BC). However, it remains under-utilized due to lack of standardized robust measurement methodologies and significant intratumor heterogeneity of expression. A recently proposed methodology for IHC biomarker assessment in whole slide images (WSI), based on systematic subsampling of tissue information extracted by digital image analysis (DIA) into hexagonal tiling arrays, enables computation of a comprehensive set of Ki67 indicators, including intratumor variability. In this study, the tiling methodology was applied to assess Ki67 expression in WSI of 152 surgically removed Ki67-stained (on full-face sections) BC specimens and to test which, if any, Ki67 indicators can predict overall survival (OS). Visual Ki67 IHC estimates and conventional clinico-pathologic parameters were also included in the study. Analysis revealed linearly independent intrinsic factors of the Ki67 IHC variance: proliferation (level of expression), disordered texture (entropy), tumor size and Nottingham Prognostic Index, bimodality, and correlation. All visual and DIA-generated indicators of the level of Ki67 expression provided significant cutoff values as single predictors of OS. However, only bimodality indicators (Ashman's D, in particular) were independent predictors of OS in the context of hormone receptor and HER2 status. From this, we conclude that spatial heterogeneity of proliferative tumor activity, measured by DIA of Ki67 IHC expression and analyzed by the hexagonal tiling approach, can serve as an independent prognostic indicator of OS in BC patients that outperforms the prognostic power of the level of proliferative activity. PMID:26818835

  19. Synthesis H-Zeolite catalyst by impregnation KI/KIO3 and performance test catalyst for biodiesel production

    NASA Astrophysics Data System (ADS)

    Widayat, W.; Rizky Wicaksono, Adit; Hakim Firdaus, Lukman; Okvitarini, Ndaru

    2016-02-01

    The objective of this research is to produce H-catalyst catalyst that was impregnated with KI/KIO3. The catalyst was analyzed about surface area, X-Ray Diffraction (XRD) and performance test of catalyst for biodiesel production. An H-Zeolite catalyst was synthesized from natural zeolite with chemical treatment processing, impregnation KI/KIO3 and physical treatment. The results shows that the surface area of the catalyst by 27.236 m2/g at a concentration of 5% KI. XRD analysis shows peak 2-θ at 23.627o indicating that KI was impregnated on H-zeolite catalyst. The catalyst was tested in production of biodiesel using palm oil with conventional methods for 3 hour at temperature of 70-80 oC. The result for conversion Fatty Acid Methyl Ester (FAME) reached maximum value on 87.91% under production process using catalyst 5% KIO3-H zeolite.

  20. Rapid Determination of the Specificity Constant of Irreversible Inhibitors (kinact/KI) by Means of an Endpoint Competition Assay.

    PubMed

    Miyahisa, Ikuo; Sameshima, Tomoya; Hixon, Mark S

    2015-11-16

    Owing to their covalent target occupancy, irreversible inhibitors require low exposures and offer long duration, and their use thus represents a powerful strategy for achieving pharmacological efficacy. Importantly, the potency metric of irreversible inhibitors is kinact/KI not IC50. A simple approach to measuring kinact/KI was developed that makes use of an irreversible probe for competitive assays run to completion against test compounds. In this system, the kinact/KI value of the test compound is equal to (kinact/KI)probe ×[probe]/IC50. The advantages of this method include simplicity, high throughput, and application to all target classes, and it only requires an in-depth kinetic evaluation of the probe.

  1. Morphometric analysis of Ki-67 and p16 expression in laryngeal precursor lesions.

    PubMed

    Pavlovic, Bojan; Djukic, Vojko; Milovanovic, Jovica; Tomanovic, Nada; Milovanovic, Aleksandar; Trivic, Aleksandar

    2013-03-01

    Laryngeal precursor lesions represent areas of altered epithelium with an increased likelihood for progression to squamous cell carcinoma. The exact molecular mechanisms of malignant transformation of laryngeal mucosa are not completely clear, but are certainly due to deregulation of cell proliferation. To assess the potential value of the p16 and Ki-67 as markers of malignant progression, we undertook a retrospective immunohistochemical and morphometric analysis on biopsy specimens from patients with precancerous lesions in the larynx. Morphometric analysis of samples stained with p16 antibody showed epithelial cell positivity in 29 (100 %) of samples with simple hyperplasia, 31 (100 %) samples with basal/parabasal cell hyperplasia, 23 (88 %) samples with atypical hyperplasia and 20 (95 %) samples with in situ carcinoma. There was a significant difference in percentage of p16-positive cells between samples with simple hyperplasia and samples with in situ carcinoma. Morphometric analysis of samples stained with Ki-67 antibody showed epithelial cell positivity in 27 (93 %) of samples with simple hyperplasia, 30 (97 %) samples with basal/parabasal cell hyperplasia, 26 (100 %) samples with atypical hyperplasia and 18 (86 %) samples with in situ carcinoma. There was a significant difference not only in the percentage of Ki-67-positive cells between samples with simple hyperplasia and samples with in situ carcinoma, but also between samples with simple and basal/parabasal cell hyperplasia. Laryngeal epithelial precursor lesions show significantly opposite patterns in p16 and Ki-67 immunopositivity. Simple hyperplasia on average shows 12 % of Ki-67-positive cells and 46 % of p16-positive cells. In situ carcinoma on average shows 23 % of Ki-67-positive cells and 36 % of p16-positive cells. PMID:23408022

  2. Ki-67 proliferation index but not mitotic thresholds integrates the molecular prognostic stratification of lower grade gliomas

    PubMed Central

    Duregon, Eleonora; Bertero, Luca; Pittaro, Alessandra; Soffietti, Riccardo; Rudà, Roberta; Trevisan, Morena; Papotti, Mauro; Ventura, Laura; Senetta, Rebecca; Cassoni, Paola

    2016-01-01

    Despite several molecular signatures for “lower grade diffuse gliomas” (LGG) have been identified, WHO grade still remains a cornerstone of treatment guidelines. Mitotic count bears a crucial role in its definition, although limited by the poor reproducibility of standard Hematoxylin & Eosin (H&E) evaluation. Phospho-histone-H3 (PHH3) and Ki-67 have been proposed as alternative assays of cellular proliferation. Therefore in the present series of 141 LGG, the molecular characterization (namely IDH status, 1p/19q co-deletion and MGMT promoter methylation) was integrated with the tumor “proliferative trait” (conventional H&E or PHH3-guided mitotic count and Ki-67 index) in term of prognosis definition. Exclusively high PHH3 and Ki-67 values were predictor of poor prognosis (log rank test, P = 0.0281 for PHH3 and P = 0.032 for Ki-67), unlike standard mitotic count. Based on Cox proportional hazard regression analyses, among all clinical (age), pathological (PHH3 and Ki-67) and molecular variables (IDH, 1p/19q codeletion and MGMT methylation) with a prognostic relevance at univariate survival analysis, only IDH expression (P = 0.001) and Ki-67 proliferation index (P = 0.027) proved to be independent prognostic factors. In addition, stratifying by IDH expression status, high Ki-67 retained its prognostic relevance uniquely in the IDH negative patient (P = 0.029) doubling their risk of death (hazard ratio = 2.27). Overall, PHH3 immunostaining is the sole reliable method with a prognostic value to highlight mitotic figures in LGG. Ki-67 proliferation index exceeds PHH3 mitotic count as a predictor of patient's prognosis, and should be integrated with molecular markers in a comprehensive grading system for LGG. PMID:27049832

  3. Ki-67 proliferation index but not mitotic thresholds integrates the molecular prognostic stratification of lower grade gliomas.

    PubMed

    Duregon, Eleonora; Bertero, Luca; Pittaro, Alessandra; Soffietti, Riccardo; Rudà, Roberta; Trevisan, Morena; Papotti, Mauro; Ventura, Laura; Senetta, Rebecca; Cassoni, Paola

    2016-04-19

    Despite several molecular signatures for "lower grade diffuse gliomas" (LGG) have been identified, WHO grade still remains a cornerstone of treatment guidelines. Mitotic count bears a crucial role in its definition, although limited by the poor reproducibility of standard Hematoxylin & Eosin (H&E) evaluation. Phospho-histone-H3 (PHH3) and Ki-67 have been proposed as alternative assays of cellular proliferation. Therefore in the present series of 141 LGG, the molecular characterization (namely IDH status, 1p/19q co-deletion and MGMT promoter methylation) was integrated with the tumor "proliferative trait" (conventional H&E or PHH3-guided mitotic count and Ki-67 index) in term of prognosis definition. Exclusively high PHH3 and Ki-67 values were predictor of poor prognosis (log rank test, P = 0.0281 for PHH3 and P = 0.032 for Ki-67), unlike standard mitotic count. Based on Cox proportional hazard regression analyses, among all clinical (age), pathological (PHH3 and Ki-67) and molecular variables (IDH, 1p/19q codeletion and MGMT methylation) with a prognostic relevance at univariate survival analysis, only IDH expression (P = 0.001) and Ki-67 proliferation index (P = 0.027) proved to be independent prognostic factors. In addition, stratifying by IDH expression status, high Ki-67 retained its prognostic relevance uniquely in the IDH negative patient (P = 0.029) doubling their risk of death (hazard ratio = 2.27). Overall, PHH3 immunostaining is the sole reliable method with a prognostic value to highlight mitotic figures in LGG. Ki-67 proliferation index exceeds PHH3 mitotic count as a predictor of patient's prognosis, and should be integrated with molecular markers in a comprehensive grading system for LGG. PMID:27049832

  4. Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity

    SciTech Connect

    Francoeur, A.M.; Peebles, C.L.; Gompper, P.T.; Tan, E.M.

    1986-03-01

    Anti-Ki (Ku, p70/p80) autoantibodies, named after the prototype patient Kikuta by Tojo et al., occur in approximately 10% of patients with SLE, often in association with anti-Sm autoantibodies. Anti-Ki sera specifically immunoprecipitated two protein antigens, Ki/sub 86/ (M/sub r/ 86,000) and Ki/sub 66/ (M/sub r/ 66,000), from radiolabeled cell extracts. The Ki system was found to be immunologically identical to the Ku system described by Mimori et al. and the p70/p80 system described by Reeves. The Ki primary in vitro translation products were identified and proved similar in size to the cellular antigens. The Ki antigens were purified from human spleen by immunoaffinity chromatography followed by SDS-PAGE. The purified Ki antigens proved to be closely related by amino acid composition, and did not appear to be phosphorylated, glycosylated, or associated with RNA. The Ki antigens were found to bind to DNA, in agreement with the observations on the Ku and p70/p80 antigens. They were found to be widely conserved in mammals and were coordinately expressed in all tissues tested.

  5. Relative binding affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy.

    PubMed

    Leu, Chih-Tai; Luegmayr, Eva; Freedman, Leonard P; Rodan, Gideon A; Reszka, Alfred A

    2006-05-01

    Potent bisphosphonates (BPs) preferentially bind bone at sites of active osteoclastic bone resorption, where they are taken up by the osteoclast and inhibit resorption. We tested the hypothesis that BP affinity to human bone affects antiresorptive potency. [(1)(4)C]-Alendronate binding to human bone was saturable and reversible with an apparent Kd of 72 microM by Scatchard analysis. In competition binding assays, unlabeled alendronate (Ki: 61 microM) was slightly more potent than pyrophosphate (Ki = 156 microM) in blocking [(1)(4)C]-alendronate binding. Likewise, most tested BPs, including etidronate (Ki: 91 microM), ibandronate (116 microM), pamidronate (83 microM), risedronate (85 microM) and zoledronate (81 microM), showed comparable affinities. Interestingly, tiludronate (173 microM; P < 0.05 vs. all other BPs) and especially clodronate (806 microM; P > 0.0001 vs. all other BPs) displayed significantly weaker affinity for bone. The weak affinity of clodronate translated into a requirement for 10-fold higher dosing in in vitro bone resorption assays when bone was pretreated with BP and subsequently washed prior to adding osteoclasts. In stark contrast, neither alendronate nor risedronate lost any efficacy after washing the bone surface. These findings suggest that most clinically tested BPs may have similar affinities for human bone. For those with reduced affinity, this may translate into lower potency that necessitates higher dosing.

  6. Affine projective Osserman structures

    NASA Astrophysics Data System (ADS)

    Gilkey, P.; Nikčević, S.

    2013-08-01

    By considering the projectivized spectrum of the Jacobi operator, we introduce the concept of projective Osserman manifold in both the affine and in the pseudo-Riemannian settings. If M is an affine projective Osserman manifold, then the deformed Riemannian extension metric on the cotangent bundle is both spacelike and timelike projective Osserman. Since any rank-1-symmetric space is affine projective Osserman, this provides additional information concerning the cotangent bundle of a rank-1 Riemannian symmetric space with the deformed Riemannian extension metric. We construct other examples of affine projective Osserman manifolds where the Ricci tensor is not symmetric and thus the connection in question is not the Levi-Civita connection of any metric. If the dimension is odd, we use methods of algebraic topology to show the Jacobi operator of an affine projective Osserman manifold has only one non-zero eigenvalue and that eigenvalue is real.

  7. Translation, cultural adaptation and validation of the Kidney Disease Knowledge Survey (KiKS) to Spanish

    PubMed Central

    Anaya, Evelin Mota; Wright Nunes, Julie A.; Mayta- Tristán, Percy

    2016-01-01

    Introduction Chronic kidney disease (CKD) affects 50 million people globally. Several studies show the importance of implementing interventions that enhance patients' knowledge about their disease. In 2011, the Kidney Disease Knowledge Survey (KiKS) was developed, a questionnaire that assesses the specific knowledge about CKD in pre-dialysis patients. Objective To translate to Spanish, culturally adapt and validate the questionnaire KiKS in a population of patients with pre-dialysis CKD. Methods The translation and cultural adaptation of KiKS was performed. Subsequently, its validity and reliability were determined. The validity was evaluated by construct validity; and the reliability by its internal consistency and its intra-observer reliability (test-retest). Results A good internal consistency was found (Kuder-Richardson = 0.85). Regarding intra-observer reliability, the intraclass correlation coefficient with a value of 0.78 (95% CI: 0.5–1.0) indicated a good reproducibility; the mean difference of −1.1 test-retest S.D. 6.0 (p = 0.369) confirm this. Conclusions The Spanish version of KiKS is acceptable and equivalent to the original version and has good reliability, validity and reproducibility. Therefore, it could be used in a population of culturally similar patients with pre-dialysis CKD. PMID:27513762

  8. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  9. The ACE inhibitor ( sup 3 H)SQ29,852 identifies a high affinity recognition site located in the human temporal cortex

    SciTech Connect

    Barnes, N.M.; Costall, B.; Egli, P.; Horovitz, Z.P.; Ironside, J.W.; Naylor, R.J.; Williams, T.J. )

    1990-07-01

    The angiotensin converting enzyme (ACE) inhibitor ({sup 3}H)SQ29,852 identified a single high affinity recognition site (defined by 10.0 microM captopril) in the human temporal cortex (pKD 8.62 +/- 0.03; Bmax 248 +/- 24 fmol mg-1 protein, mean +/- S.E.M., n = 4). ACE inhibitors and thiorphan competed to a similar level for the ({sup 3}H)SQ29,852 binding site in the human temporal cortex with a rank order of affinity (pKi values mean +/- S.E.M., n = 3), lisinopril (9.49 +/- 0.02), captopril (9.16 +/- 0.08), SQ29,852 (8.58 +/- 0.04), epicaptopril (7.09 +/- 0.08), fosinopril (7.08 +/- 0.05) and thiorphan (6.40 +/- 0.04). Since this rank order of affinity is similar to the affinity of these compounds to inhibit brain ACE activity it is concluded that ({sup 3}H)SQ29,852 selectively labels the inhibitor recognition site of ACE in the human temporal cortex.

  10. The adenosine receptor affinities and monoamine oxidase B inhibitory properties of sulfanylphthalimide analogues.

    PubMed

    Van der Walt, Mietha M; Terre'Blanche, Gisella; Petzer, Anél; Petzer, Jacobus P

    2015-04-01

    Based on a report that sulfanylphthalimides are highly potent monoamine oxidase (MAO) B selective inhibitors, the present study examines the adenosine receptor affinities and MAO-B inhibitory properties of a series of 4- and 5-sulfanylphthalimide analogues. Since adenosine antagonists (A1 and A2A subtypes) and MAO-B inhibitors are considered agents for the therapy of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs that antagonize adenosine receptors and inhibit MAO-B may have enhanced therapeutic value. The results document that the sulfanylphthalimide analogues are selective for the adenosine A1 receptor over the A2A receptor subtype, with a number of compounds also possessing MAO-B inhibitory properties. Among the compounds evaluated, 5-[(4-methoxybenzyl)sulfanyl]phthalimide was found to possess the highest binding affinity to adenosine A1 receptors with a Ki value of 0.369 μM. This compound is reported to also inhibit MAO-B with an IC50 value of 0.020 μM. Such dual-target-directed compounds may act synergistic in the treatment of Parkinson's disease: antagonism of the A1 receptor may facilitate dopamine release, while MAO-B inhibition may reduce dopamine metabolism. Additionally, dual-target-directed compounds may find therapeutic value in Alzheimer's disease: antagonism of the A1 receptor may be beneficial in the treatment of cognitive dysfunction, while MAO-B inhibition may exhibit neuroprotective properties. In neurological diseases, such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs are expected to be advantageous over single-target treatments.

  11. Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl.

    PubMed Central

    Murthy, J. V.; Kim, H. H.; Hanesworth, V. R.; Hugdahl, J. D.; Morejohn, L. C.

    1994-01-01

    Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicotiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds [14C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (Kd) = 117 nM (pH 6.9; 23[deg]C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharamacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of [14C]oryzalin to tubulin with an inhibition constant (Ki) = 5 [mu]M, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition is caused by microtubules depolymerization. APM had no apparent effect on microtubules in mouse 3T3 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a

  12. Engineering Lower Inhibitor Affinities in Beta-D-Xylosidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta- xylosidase catalyzes hydrolysis of xylooligosaccharides to D-xylose residues. The enzyme, SXA from Selenomonas ruminantium is the most active catalyst known for the reaction; however, its activity is inhibited by D-xylose and D-glucose (Ki values of ~10-5 M). Higher Ki’s could enhance enzyme...

  13. The proliferation marker Ki67, but not neuroendocrine expression, is an independent factor in the prediction of prognosis of primary prostate cancer patients

    PubMed Central

    Pascale, Mariarosa; Aversa, Cinzia; Barbazza, Renzo; Marongiu, Barbara; Siracusano, Salvatore; Stoffel, Flavio; Sulfaro, Sando; Roggero, Enrico; Stanta, Giorgio

    2016-01-01

    Abstract Background Neuroendocrine markers, which could indicate for aggressive variants of prostate cancer and Ki67 (a well-known marker in oncology for defining tumor proliferation), have already been associated with clinical outcome in prostate cancer. The aim of this study was to investigate the prognostic value of those markers in primary prostate cancer patients. Patients and methods NSE (neuron specific enolase), ChrA (chromogranin A), Syp (Synaptophysin) and Ki67 staining were performed by immunohistochemistry. Then, the prognostic impact of their expression on overall survival was investigated in 166 primary prostate cancer patients by univariate and multivariate analyses. Results NSE, ChrA, Syp and Ki67 were positive in 50, 45, 54 and 146 out of 166 patients, respectively. In Kaplan-Meier analysis only diffuse NSE staining (negative vs diffuse, p = 0.004) and Ki67 (≤ 10% vs > 10%, p < 0.0001) were significantly associated with overall survival. Ki67 expression, but not NSE, resulted as an independent prognostic factor for overall survival in multivariate analysis. Conclusions A prognostic model incorporating Ki67 expression with clinical-pathological covariates could provide additional prognostic information. Ki67 may thus improve prediction of prostate cancer outcome based on standard clinical-pathological parameters improving prognosis and management of prostate cancer patients.

  14. The proliferation marker Ki67, but not neuroendocrine expression, is an independent factor in the prediction of prognosis of primary prostate cancer patients

    PubMed Central

    Pascale, Mariarosa; Aversa, Cinzia; Barbazza, Renzo; Marongiu, Barbara; Siracusano, Salvatore; Stoffel, Flavio; Sulfaro, Sando; Roggero, Enrico; Stanta, Giorgio

    2016-01-01

    Abstract Background Neuroendocrine markers, which could indicate for aggressive variants of prostate cancer and Ki67 (a well-known marker in oncology for defining tumor proliferation), have already been associated with clinical outcome in prostate cancer. The aim of this study was to investigate the prognostic value of those markers in primary prostate cancer patients. Patients and methods NSE (neuron specific enolase), ChrA (chromogranin A), Syp (Synaptophysin) and Ki67 staining were performed by immunohistochemistry. Then, the prognostic impact of their expression on overall survival was investigated in 166 primary prostate cancer patients by univariate and multivariate analyses. Results NSE, ChrA, Syp and Ki67 were positive in 50, 45, 54 and 146 out of 166 patients, respectively. In Kaplan-Meier analysis only diffuse NSE staining (negative vs diffuse, p = 0.004) and Ki67 (≤ 10% vs > 10%, p < 0.0001) were significantly associated with overall survival. Ki67 expression, but not NSE, resulted as an independent prognostic factor for overall survival in multivariate analysis. Conclusions A prognostic model incorporating Ki67 expression with clinical-pathological covariates could provide additional prognostic information. Ki67 may thus improve prediction of prostate cancer outcome based on standard clinical-pathological parameters improving prognosis and management of prostate cancer patients. PMID:27679548

  15. Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    SciTech Connect

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C. )

    1991-02-01

    Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.

  16. Engineering lower inhibitor affinities in beta-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. One property that could use improvement is its relatively high affinities for D-glucose and D-xylose (Ki~10 mM), wh...

  17. Expressing human SHOX in Shox2SHOX KI/KI mice leads to congenital osteoarthritis-like disease of the temporomandibular joint in postnatal mice

    PubMed Central

    Liang, Wenna; Li, Xihai; Chen, Houhuang; Shao, Xiang; Lin, Xuejuan; Shen, Jianying; Ding, Shanshan; Kang, Jie; Li, Candong

    2016-01-01

    The temporomandibular joint (TMJ), a unique synovial joint whose development differs from that of other synovial joints, develops from two distinct mesenchymal condensations that grow toward each other and ossify through different mechanisms. The short stature homeobox 2 (Shox2) gene serves an important role in TMJ development and previous studies have demonstrated that Shox2SHOX KI/KI mice display a TMJ defective phenotype, congenital dysplasia and premature eroding of the articular disc, which is clinically defined as a TMJ disorder. In the present study, Shox2SHOX KI/KI mouse models were used to investigate the mechanisms of congenital osteoarthritis (OA)-like disease during postnatal TMJ growth. Shox2SHOX KI/KI mice were observed to develop a severe muscle wasting syndrome from day 7 postnatal. Histological examination indicated that the condyle and glenoid fossa of Shox2SHOX KI/KI mice was reduced in size in the second week after birth. The condyles of Shox2SHOX KI/KI mice exhibited reduced expression levels of collagen type II and Indian hedgehog, and increased expression of collagen type I. A marked increase in matrix metalloproteinase 9 (MMP9) and MMP13 in the condyles was also observed. These cellular and molecular defects may contribute to the observed (OA)-like phenotype of Shox2SHOX KI/KI mouse TMJs. PMID:27601064

  18. Affinity driven social networks

    NASA Astrophysics Data System (ADS)

    Ruyú, B.; Kuperman, M. N.

    2007-04-01

    In this work we present a model for evolving networks, where the driven force is related to the social affinity between individuals of a population. In the model, a set of individuals initially arranged on a regular ordered network and thus linked with their closest neighbors are allowed to rearrange their connections according to a dynamics closely related to that of the stable marriage problem. We show that the behavior of some topological properties of the resulting networks follows a non trivial pattern.

  19. Modification of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity labels related to peptide substrates.

    PubMed

    Bramson, H N; Thomas, N; Matsueda, R; Nelson, N C; Taylor, S S; Kaiser, E T

    1982-09-25

    The modification and concomitant inactivation of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity analogs of peptide substrates potentially capable of undergoing disulfide interchange with enzyme-bound sulfhydryl groups have been used to probe the active site associated with peptide binding. The regeneration of catalytic activity on treatment of the modified enzymes with dithiothreitol and the observation that prior reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) blocks the modification of the kinase by these reagents are consistent with the proposal that only thiol residues are reacting. The affinity analog Leu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly, 1, and the closely related peptide AcLeu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly-OEt, 3, react with a single sulfhydryl as shown by the stoichiometry of the release of the 3-nitro-2-pyridinesulfenyl group and the amount of label incorporated in the enzyme when the radioactively labeled peptide analog of 3 (peptide 4) is employed as the modifying agent. The kinetics of the reaction of 1 with 4.3 microM catalytic subunit was monophasic (employing substrate in excess conditions), yielding an apparent value of KI of approximately 40 microM and a k2 value of approximately 0.25 s-1. The low value of the observed KI, together with the observation that protein kinase substrates inhibit the modification reactions, suggest strongly that the cysteine residue undergoing reaction is in the vicinity of the active site. By trypsin-catalyzed degradation and identification of the peptide segment modified by covalent attachment of the peptide portion of the radioactive analog 4, the single cysteine modified was identified as cysteine-198.

  20. Claudin 4 expression in triple-negative breast cancer: correlation with androgen receptors and Ki-67 expression.

    PubMed

    Abd-Elazeem, Mona A; Abd-Elazeem, Marwa A

    2015-02-01

    Breast cancer is the most common malignancy in women and the leading cause of cancer mortality worldwide. Triple-negative breast cancer (TNBC) is an important phenotype of breast cancer that accounts for a relatively small number of breast cancer cases but still represent a focus of increasing interest at the clinical, biological, and epidemiological level. Claudins are the major component of the tight junction, and only a few studies have addressed the role of claudins in breast cancer, especially TNBC. Androgen receptors (ARs), as members of the nuclear receptor superfamily, are known to be involved in a complex network of signaling pathways that collectively regulate cell proliferation. However, roles of AR in breast cancer development and progression have not been very clearly understood. The proliferation marker Ki-67 has been confirmed as an independent predictive and prognostic factor in early breast cancer. The aims of this study are to identify the clinicopathologic associations and prognostic value of claudin 4 expression in TNBC and to correlate claudin 4 expression with AR status and Ki-67 expression. Paraffin blocks obtained from 56 female patients with triple-negative primary invasive ductal breast carcinomas were analyzed for claudin 4, AR, and Ki-67 immunohistochemical expression. High levels of claudin 4 expression were detected in 66.1% of TNBC cases. There was a significant positive correlation with age, tumor size, grade, nodal status, metastasis, and Ki-67 expression (all P < .05) and negative correlation with AR status (P < .001). Androgen receptor showed positivity in 29 cases (51.78%). There was a statistical negative correlation with the all the studied clinicopathologic parameters, claudin 4 and Ki-67 expression. High claudin 4 expression, negative AR expression, and high Ki-67 index would provide a strong prognostic power to differentiate the patients with worse outcome among TNBC patients. Moreover, target treatment for TNBC cells

  1. Synthesis, modelling, and mu-opioid receptor affinity of N-3(9)-arylpropenyl-N-9(3)-propionyl-3,9-diazabicycl.

    PubMed

    Pinna, G A; Murineddu, G; Curzu, M M; Villa, S; Vianello, P; Borea, P A; Gessi, S; Toma, L; Colombo, D; Cignarella, G

    2000-08-01

    A series of N-3-arylpropenyl-N-9-propionyl-3,9-diazabicyclo[3.3.1]nonanes (1a-g) and of reverted N-3-propionyl-N-9-arylpropenyl isomers (2a-g), as homologues of the previously reported analgesic 3,8-diazabicyclo[3.2.1]octanes (I-II), were synthesized and evaluated for the binding affinity towards opioid receptor subtypes mu, delta and kappa. Compounds 1a-g and 2a-g exhibited a strong selective mu-affinity with Ki values in the nanomolar range, which favourably compared with those of I and II. In addition, contrary to the trend observed for DBO-I, II, the mu-affinity of series 2 is markedly higher than that of the isomeric series 1. This aspect was discussed on the basis of the conformational studies performed on DBN which allowed hypotheses on the mode of interaction of these compounds with the mu receptor.

  2. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    SciTech Connect

    Grigoriadis, D.E.; Zaczek, R.; Pearsall, D.M.; De Souza, E.B. )

    1989-12-01

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

  3. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  4. Proton affinities of hydrated molecules

    NASA Astrophysics Data System (ADS)

    Valadbeigi, Younes

    2016-09-01

    Proton affinities (PA) of non-hydrated, M, and hydrated forms, M(H2O)1,2,3, of 20 organic molecules including alcohols, ethers, aldehydes, ketones and amines were calculated by the B3LYP/6-311++G(d,p) method. For homogeneous families, linear correlations were observed between PAs of the M(H2O)1,2,3 and the PAs of the non-hydrated molecules. Also, the absolute values of the hydration enthalpies of the protonated molecules decreased linearly with the PAs. The correlation functions predicted that for an amine with PA < 1100 kJ/mol the PA(M(H2O)) is larger than the corresponding PA, while for an amine with PA > 1100 kJ/mol the PA(M(H2O)) is smaller than the PA.

  5. Muscarinic receptors in rat nasal mucosa are predominantly of the low affinity agonist type.

    PubMed

    Rodrigues de Miranda, J F; Scheres, H M; Salden, H J; Beld, A J; Klaassen, A B; Kuijpers, W

    1985-07-31

    Specific [3H]l-quinuclidinyl benzilate binding to rat nasal mucosa homogenates occurs to a homogeneous class of binding sites with Kd = 60 +/- 2 10(-12) M and Bmax = 8.1 +/- 2 pmol/g tissue. Binding is stereoselectively inhibited by benzetimide enantiomers. Pirenzepine inhibits [3H]l-quinuclidinyl benzilate binding with low affinity (Ki = 5.0 10(-7) M), classifying the binding sites as muscarinic M2-receptors. Methylfurtrethonium and methacholine inhibit [3H]l-quinuclidinyl benzilate binding following an almost sigmoid curve at high concentrations pointing to the presence of mainly low affinity agonist binding sites. PMID:3840092

  6. [Neuroendocrine tumors of digestive system: morphologic spectrum and cell proliferation (Ki67 index)].

    PubMed

    Delektorskaia, V V; Kushliskiĭ, N E

    2013-01-01

    This review deals with the analysis of up-to-date concepts ofdiferent types of human neuroendocrine tumors of the digestive system. It summarizes the information on the specifics of recent histological classifications and criteria of morphological diagnosis accounting histological, ultrastructural and immunohistochemical parameters. Current issues of the nomenclature as well as various systems of grading and staging are discussed. In the light of these criteria the results of the own research clinical value of the determination of cell proliferation in primary and metastatic gastroenteropancreatic neuroendocrine neoplasms on the basis of evaluation of the Ki67 antigen expression are also presented.

  7. Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II alpha. Delineation of a carboxy-terminal epitope with peptide antibodies.

    PubMed Central

    Boege, F.; Andersen, A.; Jensen, S.; Zeidler, R.; Kreipe, H.

    1995-01-01

    Proliferation-linked expression of the nuclear Ki-S1 antigen is a significant prognostic indicator in mammary carcinomas. Here, we show staining of a protein of 170 kd by Ki-S1 antibody in immunoblots of Saccharomyces cerevisiae expressing human topoisomerase II alpha but not in the parental strain. In HL-60 cells containing both isoforms of human topoisomerase II, Ki-S1 antibody binds selectively to the 170-kd isoenzyme in a similar fashion as peptide-antibodies directed against amino acid residues 1 to 15 or 1512 to 1530 of human topoisomerase II alpha. Conversely, antibodies directed against carboxyl-terminal sequences of human topoisomerase II beta selectively stain a 180-kd protein. The immunoreactive pattern of V8 endoproteinase restriction digests of human topoisomerase II alpha was identical for Ki-S1-antibody and peptide-antibodies directed against residues 1512 to 1530 but different for peptide-antibodies directed against residues 1 to 15. The Rf values of the smallest fragment commonly recognized by Ki-S1 antibody and the carboxy terminus-specific peptide-antibody place the Ki-S1 epitope within the last 495 carboxyl-terminal amino acid residues of topoisomerase II alpha. Images Figure 1 Figure 2 Figure 3 PMID:7539979

  8. Immunohistochemistry and scoring of Ki-67 proliferative index and p53 expression in gastric B cell lymphoma from Northern African population: a pilot study

    PubMed Central

    Zeggai, Soumia; Tou, Abdelnacer; Sellam, Feriel; Mrabent, Meriem N.; Salah, Rachida

    2016-01-01

    Background This study aimed to clarify the Ki-67 distribution, p53 expression and their relationship with clinico-pathologic features of gastric B cell lymphoma from Northern African population. Methods Twenty paraffin blocks of gastric lymphoma were retrieved from the archival materials of Department of Pathology, Central University Hospital of Sidi Bel Abbes (Western Algeria) from 2007 to 2013. Four µm section specimens were stained by immunohistochemical (IHC) technique with Ki-67 and p53 tumor markers. P values <0.05 were considered statistically significant. Results Expression of p53 proteins and the mean proliferative index (PI) were compared between high grade gastric B cell lymphomas (DLBCL) and low grade gastric B cell lymphomas (gastric MALTs). p53 overexpression (P=0.007) and a high proliferation index Ki-67 (P=0.001) were significantly associated with gastric DLBCL. We found also a statistically significant correlation between p53 and Ki-67 (P=0.007) but no obvious relationships were found between Ki-67 PI and p53 expression as well as clinico-pathological features (age, sex, location, macroscopic type). Conclusions The IHC studies of Ki-67 and p53 expression in gastric B cell lymphoma can help in monitoring of patients at risk, and to give suitable treatment and management of patients. PMID:27284480

  9. Association of mammographic density with the proliferation marker Ki-67 in a cohort of patients with invasive breast cancer.

    PubMed

    Heusinger, Katharina; Jud, Sebastian M; Häberle, Lothar; Hack, Carolin C; Fasching, Peter A; Meier-Meitinger, Martina; Lux, Michael P; Hagenbeck, Carsten; Loehberg, Christian R; Wittenberg, Thomas; Rauh, Claudia; Wagner, Florian; Uder, Michael; Hartmann, Arndt; Schulz-Wendtland, Rüdiger; Beckmann, Matthias W; Wachter, David L

    2012-10-01

    There is growing evidence that certain breast cancer (BC) risk factors specifically increase the risk for specific molecular tumor subtypes. Different molecular subtypes of BC can partly be described by analyzing proliferation in tumors. Very few data are available regarding the association of mammographic density (MD), as a BC risk factor, with proliferation. The aim of this study was to analyze the association between Ki-67 expression in BCs and MD. In this case-only study, data on BC risk factors, hormone receptor expression, and MD were available for 1,975 patients with incident BC. MD was assessed as percentage mammographic density (PMD) using a semiautomated method by two readers for every patient. The association of the Ki-67 proliferation index and PMD was studied using multifactorial analyses of covariance (ANCOVA), with PMD as the target variable and including well-known factors that are also associated with MD such as age, parity, use of hormone replacement therapy (HRT), and body mass index (BMI). There were no significant differences in PMD between women with BC who had low and high Ki-67 values (P = 0.31). However, there were relevant differences in women with low BMI (P = 0.07), and in women using postmenopausal HRT (P = 0.06) as well as in women with low PR values (P = 0.07). In these subgroups, the Ki-67 expression index increased with decreasing PMD. Likewise PMD is correlated with BMI, parity status, and menopausal status stronger in patients with low proliferating tumors, and with progesterone receptor expression in patients with high proliferating tumors. MD correlates inversely with Ki-67 proliferation in BC tumors only in some subgroups of BC patients, defined by commonly known BC risk factors that are usually associated with MD as well.

  10. Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat.

    PubMed

    Navarro, V M; Castellano, J M; Fernández-Fernández, R; Tovar, S; Roa, J; Mayen, A; Barreiro, M L; Casanueva, F F; Aguilar, E; Dieguez, C; Pinilla, L; Tena-Sempere, M

    2005-04-01

    KiSS-1 was originally identified as a metastasis suppressor gene encoding an array of structurally related peptides, namely kisspeptins, which acting through the G protein-coupled receptor GPR54 are able to inhibit tumor progression. Unexpectedly, a reproductive facet of this newly discovered system has recently arisen, and characterization of the role of the KiSS-1/GPR54 system in the neuroendocrine control of gonadotropin secretion has been initiated. However, such studies have been so far mostly restricted to LH, and very little is known about the actual contribution of this system in the regulation of FSH release. To address this issue, the effects of KiSS-1 peptide on FSH secretion were monitored in vivo and in vitro under different experimental conditions. Intracerebroventricular administration of KiSS-1 peptide significantly stimulated FSH secretion in prepubertal and adult rats. Yet, dose-response analyses in vivo demonstrated an ED(50) value for the FSH-releasing effects of KiSS-1 of 400 pmol, i.e. approximately 100-fold higher than that of LH. In addition, systemic (ip and iv) injection of KiSS-1 significantly stimulated FSH secretion in vivo. However, KiSS-1 failed to elicit basal FSH release directly at the pituitary level, although it moderately enhanced GnRH-stimulated FSH secretion in vitro. Finally, mechanistic studies revealed that the ability of KiSS-1 to elicit FSH secretion was abolished by the blockade of endogenous GnRH actions, but it was persistently observed in different models of leptin insufficiency and after blockade of endogenous excitatory amino acid and nitric oxide pathways, i.e. relevant signals in the neuroendocrine control of gonadotropin secretion. In summary, our results extend previous recent observations on the role of KiSS-1 in the control of LH secretion and provide solid evidence for a stimulatory effect of KiSS-1 on FSH release, acting at central level. Overall, it is proposed that the KiSS-1/GPR54 system is a novel

  11. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  12. Expression of MTA2 and Ki-67 in hepatocellular carcinoma and their correlation with prognosis

    PubMed Central

    Shi, Wei; Hu, Jiangfeng; Zhu, Shizhang; Shen, Xiaoying; Zhang, Xiaoyan; Yang, Changqing; Gao, Hengjun; Zhang, Hao

    2015-01-01

    The aim of this study was to investigate the relationship among MTA2, Ki-67 and HCC patient prognosis. Tissue microarray and immunohistochemistry were used to detect the expression of MTA2 and Ki-67 in HCC samples and corresponding adjacent samples. We found MTA2 and Ki-67 were both increased in HCC tissues than those in adjacent tissues and nuclear MTA2 was associated with Ki-67 (P = 0.019). Moreover, nuclear MTA2 was a risk factor of distant metastasis in patients with HCC andKi-67 showed a negative correlation with histological grade (P < 0.05, P < 0.01, respectively). Multivariate Cox model analysis revealed that Ki-67 expression was an independent prognosis factor in HCC patients (P = 0.020). These results indicated there might be a tight correlation among MTA2, Ki-67 and HCC prognosis. MTA2 combined with Ki-67 might be used to predict HCC patient prognosis. PMID:26722504

  13. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  14. Distinguishing Low-Risk Luminal A Breast Cancer Subtypes with Ki-67 and p53 Is More Predictive of Long-Term Survival.

    PubMed

    Lee, Se Kyung; Bae, Soo Youn; Lee, Jun Ho; Lee, Hyun-Chul; Yi, Hawoo; Kil, Won Ho; Lee, Jeong Eon; Kim, Seok Won; Nam, Seok Jin

    2015-01-01

    Overexpression of p53 is the most frequent genetic alteration in breast cancer. Recently, many studies have shown that the expression of mutant p53 differs for each subtype of breast cancer and is associated with different prognoses. In this study, we aimed to determine the suitable cut-off value to predict the clinical outcome of p53 overexpression and its usefulness as a prognostic factor in each subtype of breast cancer, especially in luminal A breast cancer. Approval was granted by the Institutional Review Board of Samsung Medical Center. We analyzed a total of 7,739 patients who were surgically treated for invasive breast cancer at Samsung Medical Center between Dec 1995 and Apr 2013. Luminal A subtype was defined as ER&PR + and HER2- and was further subclassified according to Ki-67 and p53 expression as follows: luminal A (Ki-67-,p53-), luminal A (Ki-67+, p53-), luminal A (Ki-67 -, p53+) and luminal A (Ki-67+, p53+). Low-risk luminal A subtype was defined as negative for both Ki-67 and p53 (luminal A [ki-67-, p53-]), and others subtypes were considered to be high-risk luminal A breast cancer. A cut-off value of 10% for p53 was a good predictor of clinical outcome in all patients and luminal A breast cancer patients. The prognostic role of p53 overexpression for OS and DFS was only significant in luminal A subtype. The combination of p53 and Ki-67 has been shown to have the best predictive power as calculated by the area under curve (AUC), especially for long-term overall survival. In this study, we have shown that overexpression of p53 and Ki-67 could be used to discriminate low-risk luminal A subtype in breast cancer. Therefore, using the combination of p53 and Ki-67 expression in discriminating low-risk luminal A breast cancer may improve the prognostic power and provide the greatest clinical utility.

  15. Vincristine modulates the expression of Ki67 and apoptosis in naturally occurring canine transmissible venereal tumor (TVT).

    PubMed

    Özalp, G R; Zik, B; Bastan, A; Peker, S; Özdemir-Salci, E S; Bastan, I; Darbaz, I; Salar, S; Karakas, K

    2012-07-01

    We investigated eight adult dogs that were brought to veterinary clinics with a history of transmissible venereal tumors (TVT). Our goal was to demonstrate the occurrence of apoptosis and the cessation of cell proliferation at every phase of scheduled chemotherapy for naturally occurring TVT. Tissue samples were collected immediately after weekly treatments with vincristine sulfate and processed for histological purposes. Sections 5 μm thick were stained by the TUNEL reaction for apoptosis and immunostained for Ki67 as a proliferation marker. We observed that after vincristine applications, tumor cell proliferation ceased and apoptosis increased. Ki67 HSCORE values were significantly lowered after the first and second treatments with the chemotherapeutic agent compared to controls, whereas TUNEL HSCORE values were significantly higher after two applications of vincristine compared to controls. Our results suggest that scheduled vincristine sulfate applications stabilize the induction of tumor regression by inducing apoptosis and preventing cell proliferation.

  16. Ki: A Key to Transform the Century of Death to the Century of Life

    PubMed Central

    2007-01-01

    This is my response to the commentary written by Mr James Flowers with the title of ‘What is Qi?’ in the issue 4 of Vol.3 (2006) of eCAM. I will explain my opinions regarding the importance of Ki research, philosophical aspects of Ki and a possible role of Ki now and in the future. PMID:17965758

  17. Evaluation of Ki67, p16 and CK17 Markers in Differentiating Cervical Intraepithelial Neoplasia and Benign Lesions

    PubMed Central

    Sari Aslani, Fatemeh; Safaei, Akbar; Pourjabali, Masoumeh; Momtahan, Mozhdeh

    2013-01-01

    Background: Cervical intraepithelial neoplasia (CIN) is a premalignant lesion capable of progressing to cervical cancer. Despite the existing well-defined criteria, the histomorphologic diagnosis is subject to high rates of discordance among pathologists. The aim of this study was to evaluate Ki-67 (MIB-1), CK17 and p16 INK4a (p16) markers by immunohistochemical methods in differentiating CIN from benign cervical lesions. Methods: The present study reviewed and re-classified 77 cervical biopsies, originally diagnosed as 31 non-CIN, and 46 CIN, as 54 non-CIN, and 23 CIN based on at least two similar diagnoses. Immunostaining by Ki67, p16 and CK17 markers was performed on all cases and the results were compared with pervious and consensus diagnosis. Results: The overall agreement between pervious and consensus diagnosis was 67.5% (Kappa=0.39, P<0.001). The sensitivity and specificity of Ki67 immunostaining were 95.6% and 85.1% respectively, while for p16 the corresponding values were 91.3% and 98.1%. The overall agreement, for both p16 and Ki67, with consensus diagnosis were significant (P<0.001). The sensitivity and specificity of CK17 negative staining in CIN detection were 39.1% and 40.7% respectively. Conclusion: Ki67 and p16 markers are recommended as complementary tests for differentiating between dysplastic and non-dysplastic lesions. CK17 does not discriminate between immature metaplasia with and without dysplasia. PMID:23645953

  18. High‐throughput automated scoring of Ki67 in breast cancer tissue microarrays from the Breast Cancer Association Consortium

    PubMed Central

    Howat, William J; Daley, Frances; Zabaglo, Lila; McDuffus, Leigh‐Anne; Blows, Fiona; Coulson, Penny; Raza Ali, H; Benitez, Javier; Milne, Roger; Brenner, Herman; Stegmaier, Christa; Mannermaa, Arto; Chang‐Claude, Jenny; Rudolph, Anja; Sinn, Peter; Couch, Fergus J; Tollenaar, Rob A.E.M.; Devilee, Peter; Figueroa, Jonine; Sherman, Mark E; Lissowska, Jolanta; Hewitt, Stephen; Eccles, Diana; Hooning, Maartje J; Hollestelle, Antoinette; WM Martens, John; HM van Deurzen, Carolien; Investigators, kConFab; Bolla, Manjeet K; Wang, Qin; Jones, Michael; Schoemaker, Minouk; Broeks, Annegien; van Leeuwen, Flora E; Van't Veer, Laura; Swerdlow, Anthony J; Orr, Nick; Dowsett, Mitch; Easton, Douglas; Schmidt, Marjanka K; Pharoah, Paul D; Garcia‐Closas, Montserrat

    2016-01-01

    Abstract Automated methods are needed to facilitate high‐throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large‐scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. We utilized 166 TMAs containing 16,953 tumour cores representing 9,059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs were stained for Ki67 using standard immunohistochemical procedures, and scanned and digitized using the Ariol system. An automated algorithm was developed for the scoring of Ki67, and scores were compared to computer assisted visual (CAV) scores in a subset of 15 TMAs in a training set. We also assessed the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, we observed good discriminatory accuracy (AUC = 85%) and good agreement (kappa = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varied by TMA (kappa range= 0.37–0.87) and study (kappa range = 0.39–0.69). The automated method performed better in satisfactory cores (kappa = 0.68) than suboptimal (kappa = 0.51) cores (p‐value for comparison = 0.005); and among cores with higher total nuclei counted by the machine (4,000–4,500 cells: kappa = 0.78) than those with lower counts (50–500 cells: kappa = 0.41; p‐value = 0.010). Among the 9,059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics were found to be in the expected directions. Our findings indicate that automated scoring of Ki67 can be an efficient method to obtain good quality data across large numbers of TMAs from multicentre studies. However, robust algorithm development and rigorous pre‐ and post

  19. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-01

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  20. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  1. Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

    PubMed Central

    Gounaris, A D; Brown, M A; Barrett, A J

    1984-01-01

    Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated. Images Fig. 2. Fig. 3. Fig. 4. PMID:6548132

  2. [Expression and clinical significance of Ki-67 in diffuse large B cell lymphoma].

    PubMed

    Zhou, Ying; Zhao, Yu; Bo, Jian; Li, Yan-Fen; Ma, Chao; Shi, Ya-Nan

    2013-10-01

    This study was aimed to evaluate the proliferation-associated antigen Ki-67 expression in diffuse large B cell lymphoma (DLBCL) and its clinical significance. The Ki-67 expression and its correlation with prognosis in 50 patients with DLBCL treated with rituximab plus CHOP (R-CHOP) between January 2008 and December 2010 were analyzed retrospectively. The results indicated that there was no significant relationship between Ki-67 expression and clinical features, including age, sex, staging, B symptoms, LDH level, IPI, extranodal site involvement, presence of bulky tumors (>10 cm in diameter), bone marrow involvement, GCG nor GCB type, or response to first line treatment. The median survival was 50 months and 15 months in low Ki-67 expression group (<85%) and in high Ki-67 expression group ( ≥ 85%) respectively. The overall survival (OS) and progress-free survival (PFS) in low Ki-67 expression group were obviously longer than that in high Ki-67 expression group (P = 0.001; P = 0.027). In univariate analysis, the clinical factors associated with OS included Ann Arbor staging and Ki-67 expression. The clinical factors associated with PFS included Ann Arbor staging. IPI and Ki-67 expression. In multivariate analysis. The Ki-67 expression level was an independent prognostic factor for OS (HR = 4.90; 95% CI, 1.456-16.511; P = 0.0103). It is concluded that Ki-67 expression level seems to be an effective marker for evaluation of DLBCL prognosis. PMID:24156426

  3. Affinity based information diffusion model in social networks

    NASA Astrophysics Data System (ADS)

    Liu, Hongli; Xie, Yun; Hu, Haibo; Chen, Zhigao

    2014-12-01

    There is a widespread intuitive sense that people prefer participating in spreading the information in which they are interested. The affinity of people with information disseminated can affect the information propagation in social networks. In this paper, we propose an information diffusion model incorporating the mechanism of affinity of people with information which considers the fitness of affinity values of people with affinity threshold of the information. We find that the final size of information diffusion is affected by affinity threshold of the information, average degree of the network and the probability of people's losing their interest in the information. We also explore the effects of other factors on information spreading by numerical simulations and find that the probabilities of people's questioning and confirming the information can affect the propagation speed, but not the final scope.

  4. Ki-67 is required for maintenance of cancer stem cells but not cell proliferation

    PubMed Central

    Cidado, Justin; Wong, Hong Yuen; Rosen, D. Marc; Cimino-Mathews, Ashley; Garay, Joseph P.; Fessler, Abigail G.; Rasheed, Zeshaan A.; Hicks, Jessica; Cochran, Rory L.; Croessmann, Sarah; Zabransky, Daniel J.; Mohseni, Morassa; Beaver, Julia A.; Chu, David; Cravero, Karen; Christenson, Eric S.; Medford, Arielle; Mattox, Austin; De Marzo, Angelo M.; Argani, Pedram; Chawla, Ajay; Hurley, Paula J.; Lauring, Josh; Park, Ben Ho

    2016-01-01

    Ki-67 expression is correlated with cell proliferation and is a prognostic marker for various cancers; however, its function is unknown. Here we demonstrate that genetic disruption of Ki-67 in human epithelial breast and colon cancer cells depletes the cancer stem cell niche. Ki-67 null cells had a proliferative disadvantage compared to wildtype controls in colony formation assays and displayed increased sensitivity to various chemotherapies. Ki-67 null cancer cells showed decreased and delayed tumor formation in xenograft assays, which was associated with a reduction in cancer stem cell markers. Immunohistochemical analyses of human breast cancers revealed that Ki-67 expression is maintained at equivalent or greater levels in metastatic sites of disease compared to matched primary tumors, suggesting that maintenance of Ki-67 expression is associated with metastatic/clonogenic potential. These results elucidate Ki-67's role in maintaining the cancer stem cell niche, which has potential diagnostic and therapeutic implications for human malignancies. PMID:26823390

  5. An international study to increase concordance in Ki67 scoring.

    PubMed

    Polley, Mei-Yin C; Leung, Samuel C Y; Gao, Dongxia; Mastropasqua, Mauro G; Zabaglo, Lila A; Bartlett, John M S; McShane, Lisa M; Enos, Rebecca A; Badve, Sunil S; Bane, Anita L; Borgquist, Signe; Fineberg, Susan; Lin, Ming-Gang; Gown, Allen M; Grabau, Dorthe; Gutierrez, Carolina; Hugh, Judith C; Moriya, Takuya; Ohi, Yasuyo; Osborne, C Kent; Penault-Llorca, Frédérique M; Piper, Tammy; Porter, Peggy L; Sakatani, Takashi; Salgado, Roberto; Starczynski, Jane; Lænkholm, Anne-Vibeke; Viale, Giuseppe; Dowsett, Mitch; Hayes, Daniel F; Nielsen, Torsten O

    2015-06-01

    Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90-0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before

  6. An international study to increase concordance in Ki67 scoring.

    PubMed

    Polley, Mei-Yin C; Leung, Samuel C Y; Gao, Dongxia; Mastropasqua, Mauro G; Zabaglo, Lila A; Bartlett, John M S; McShane, Lisa M; Enos, Rebecca A; Badve, Sunil S; Bane, Anita L; Borgquist, Signe; Fineberg, Susan; Lin, Ming-Gang; Gown, Allen M; Grabau, Dorthe; Gutierrez, Carolina; Hugh, Judith C; Moriya, Takuya; Ohi, Yasuyo; Osborne, C Kent; Penault-Llorca, Frédérique M; Piper, Tammy; Porter, Peggy L; Sakatani, Takashi; Salgado, Roberto; Starczynski, Jane; Lænkholm, Anne-Vibeke; Viale, Giuseppe; Dowsett, Mitch; Hayes, Daniel F; Nielsen, Torsten O

    2015-06-01

    Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90-0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before

  7. [Psychopathological problems and psychosocial impairment in children and adolescents aged 3-17 years in the German population: prevalence and time trends at two measurement points (2003-2006 and 2009-2012): results of the KiGGS study: first follow-up (KiGGS Wave 1)].

    PubMed

    Hölling, H; Schlack, R; Petermann, F; Ravens-Sieberer, U; Mauz, E

    2014-07-01

    Child and adolescent mental health problems burden not only the individual, but also their families and their social environment and may, therefore, be regarded as a highly relevant public health issue. The data on mental health problems of children and adolescents from the KiGGS Wave 1 study (sample period 2009-2012) make it possible to report on both current prevalence rates and time trends over the 6-year period beginning with the KiGGS baseline survey (2003-2006). The assessment of emotional and behavioral problems in KiGGS Wave 1 was carried out with the symptoms questionnaire of the Strengths and Difficulties Questionnaire (SDQ) in a telephone interview with 10,353 guardians of children and adolescents aged 3-17 years. Moreover, using the SDQ impact supplement, the KIGGS Wave 1 data provide information on psychosocial impairment following child and adolescent mental health problems. Subjects with a borderline or abnormal SDQ score, according to German normative data, were considered at risk. A total of 20.2% (95% CI: 18.9-21.6%) of the study subjects were identified as being at risk for a mental health disorder, compared with 20.0% (19.1-20.9%) during the KiGGS baseline study (age-standardized based on population from 12 December 2010). Thus, no significant changes over time in the prevalence of mental health problems were detected. Also, there were no statistically significant differences in prevalence by sex, age group, or socioeconomic status between the KiGGS baseline survey and KiGGS Wave 1. The statistical comparison of the subscale mean values for both girls and boys showed higher values with respect to the subscales for emotional problems, behavioral problems, and prosocial behavior and lower mean values for the peer problems subscale in KiGGS Wave 1. These partly small temporal trends, however, may be due to possible mode effects (written questionnaire in the KiGGS baseline study versus telephone interview in KiGGS Wave 1). The hyperactivity subscale

  8. Prognostic Utility of Apoptosis Index, Ki-67 and Survivin Expression in Dogs with Nasal Carcinoma Treated with Orthovoltage Radiation Therapy

    PubMed Central

    FU, Dah-Renn; KATO, Daiki; WATABE, Ai; ENDO, Yoshifumi; KADOSAWA, Tsuyoshi

    2014-01-01

    ABSTRACT Apoptosis, Ki-67 and survivin expression have been reported as prognostic values in human cancer treated with radiation therapy. The aim of this study was to evaluate the correlation between the outcome of canine nasal carcinomas treated with radiation therapy and these cancer markers. The apoptotic index (AI) was evaluated with TUNEL assays, and an immunohistochemical evaluation was performed on Ki-67 and survivin in 33 biopsy samples taken before treatment. Median survival times were estimated using Kaplan-Meier curves and the log-rank method. The AI ranged from 0 to 0.7%, and the percentage of Ki-67-positive cells defined as the proliferative index (PI) ranged from 0.8 to 77% in all samples. Neither the AI nor the PI had a significant relationship with survival time (P=0.056 and 0.211). Survivin expression was detected in 84.9% of samples of canine nasal carcinoma. Dogs with high survivin expression were associated with poorer response to treatment and had shorter survival times (P=0.017 and 0.031). Advanced-stage tumors were also significantly associated with a high level of survivin (P=0.026). Overexpression of survivin was shown to be an unfavorable prognostic factor in dogs with nasal carcinomas treated with radiation therapy. PMID:25452259

  9. Activated Ki-Ras complements erythropoietin signaling in CTLL-2 cells, inducing tyrosine phosphorylation of a 160-kDa protein.

    PubMed Central

    Yamamura, Y; Noda, M; Ikawa, Y

    1994-01-01

    We have previously shown that expression of erythropoietin (EPO) receptor (EPOR) alone failed to confer EPO responsiveness on the interleukin 2-dependent T-cell line CTLL-2, whereas the introduction of the EPOR into interleukin 3-dependent pro-B-cell lines, such as BAF-B03, allowed the cells to proliferate in response to EPO. Here, we report that additional expression of v-Ki-Ras conferred EPO-dependent growth on CTLL-2 cells expressing the EPOR, with additional formation of a high-affinity EPOR. To investigate possible mechanisms of EPOR downstream signaling induced by v-Ki-Ras expression in these CTLL-2-derived cells, we carried out anti-phosphotyrosine immunoblot analysis of the EPOR complex immunoprecipitated with anti-EPOR antibody from lysates of cells with and without cytokine stimulation, revealing two 160-kDa and 130-kDa phosphotyrosyl proteins. An anti-JAK2 antibody did not react with these proteins, suggesting that they may represent cellular components involved in an EPO-EPOR signaling pathway induced by v-Ki-Ras. Similar phosphotyrosyl proteins were present among Friend erythroleukemia cell lines, in which the Friend virus gp55/EPOR complex on the cell surface constitutively sends signals for cell growth. Images PMID:7522324

  10. Screening for Cervical Cancer Precursors With p16/Ki-67 Dual-Stained Cytology: Results of the PALMS Study

    PubMed Central

    2013-01-01

    Background Pap cytology is known to be more specific but less sensitive than testing for human papillomavirus (HPV) for the detection of high-grade cervical intraepithelial neoplasia (CIN2+). We assessed whether p16/Ki-67 dual-stained cytology, a biomarker combination indicative of transforming HPV infections, can provide high sensitivity for CIN2+ in screening while maintaining high specificity. Results were compared with Pap cytology and HPV testing. Methods A total of 27349 women 18 years or older attending routine cervical cancer screening were prospectively enrolled in five European countries. Pap cytology, p16/Ki-67 immunostaining, and HPV testing were performed on all women. Positive test results triggered colposcopy referral, except for women younger than 30 years with only positive HPV test results. Presence of CIN2+ on adjudicated histology was used as the reference standard. Two-sided bias-corrected McNemar P values were determined. Results The p16/Ki-67 dual-stained cytology positivity rates were comparable with the prevalence of abnormal Pap cytology results and less than 50% of the positivity rates observed for HPV testing. In women of all ages, dual-stained cytology was more sensitive than Pap cytology (86.7% vs 68.5%; P < .001) for detecting CIN2+, with comparable specificity (95.2% vs 95.4%; P = .15). The relative performance of the tests was similar in both groups of women: younger than age 30 and 30 years or older. HPV testing in women 30 years or older was more sensitive than dual-stained cytology (93.3% vs 84.7%; P = .03) but less specific (93.0% vs 96.2%; P < .001). Conclusions The p16/Ki-67 dual-stained cytology combines superior sensitivity and noninferior specificity over Pap cytology for detecting CIN2+. It suggests a potential role of dual-stained cytology in screening, especially in younger women where HPV testing has its limitations. PMID:24096620

  11. KiMoSys: a web-based repository of experimental data for KInetic MOdels of biological SYStems

    PubMed Central

    2014-01-01

    Background The kinetic modeling of biological systems is mainly composed of three steps that proceed iteratively: model building, simulation and analysis. In the first step, it is usually required to set initial metabolite concentrations, and to assign kinetic rate laws, along with estimating parameter values using kinetic data through optimization when these are not known. Although the rapid development of high-throughput methods has generated much omics data, experimentalists present only a summary of obtained results for publication, the experimental data files are not usually submitted to any public repository, or simply not available at all. In order to automatize as much as possible the steps of building kinetic models, there is a growing requirement in the systems biology community for easily exchanging data in combination with models, which represents the main motivation of KiMoSys development. Description KiMoSys is a user-friendly platform that includes a public data repository of published experimental data, containing concentration data of metabolites and enzymes and flux data. It was designed to ensure data management, storage and sharing for a wider systems biology community. This community repository offers a web-based interface and upload facility to turn available data into publicly accessible, centralized and structured-format data files. Moreover, it compiles and integrates available kinetic models associated with the data. KiMoSys also integrates some tools to facilitate the kinetic model construction process of large-scale metabolic networks, especially when the systems biologists perform computational research. Conclusions KiMoSys is a web-based system that integrates a public data and associated model(s) repository with computational tools, providing the systems biology community with a novel application facilitating data storage and sharing, thus supporting construction of ODE-based kinetic models and collaborative research projects. The web

  12. The sodium ion affinities of asparagine, glutamine, histidine and arginine

    NASA Astrophysics Data System (ADS)

    Wang, Ping; Ohanessian, Gilles; Wesdemiotis, Chrys

    2008-01-01

    The sodium ion affinities of the amino acids Asn, Gln, His and Arg have been determined by experimental and computational approaches (for Asn, His and Arg). Na+-bound heterodimers with amino acid and peptide ligands (Pep1, Pep2) were produced by electrospray ionization. From the dissociation kinetics of these Pep1-Na+-Pep2 ions to Pep1-Na+ and Pep2-Na+, determined by collisionally activated dissociation, a ladder of relative affinities was constructed and subsequently converted to absolute affinities by anchoring the relative values to known Na+ affinities. The Na+ affinities of Asn, His and Arg, were calculated at the MP2(full)/6-311+G(2d,2p)//MP2/6-31G(d) level of ab initio theory. The resulting experimental and computed Na+ affinities are in excellent agreement with one another. These results, combined with those of our previous studies, yield the sodium ion affinities of 18 out of the 20 [alpha]-amino acids naturally occurring in peptides and proteins of living systems.

  13. High affinity of sigma1-binding sites for sterol isomerization inhibitors: evidence for a pharmacological relationship with the yeast sterol C8–C7 isomerase

    PubMed Central

    Moebius, Fabian F; Reiter, Raphael J; Hanner, Markus; Glossmann, Hartmut

    1997-01-01

    The sigma-drug binding site of guinea-pig liver is carried by a protein which shares significant amino acid sequence similarities with the yeast sterol C8–C7 isomerase (ERG2 protein). Pharmacologically - but not structurally - the sigma1-site is also related to the emopamil binding protein, the mammalian sterol C8–C7 isomerase. We therefore investigated if sterol C8–C7 isomerase inhibitors are high affinity ligands for the (+)-[3H]-pentazocine labelled sigma1-binding site.Among the compounds which bound with high affinity to native hepatic and cerebral as well as to yeast expressed sigma1-binding sites were the agricultural fungicide fenpropimorph (Ki 0.005 nM), the antihypocholesterinaemic drugs triparanol (Ki 7.0 nM), AY-9944 (Ki 0.46 nM) and MDL28,815 (Ki 0.16 nM), the enantiomers of the ovulation inducer clomiphene (Ki 5.5 and 12 nM, respectively) and the antioestrogene tamoxifen (Ki 26 nM).Except for tamoxifen these affinities are essentially identical with those for the [3H]-ifenprodil labelled sterol C8–C7 isomerase of S. cerevisiae. This demonstrates that sigma1-binding protein and yeast isomerase are not only structurally but also pharmacologically related. Because of its affiliations with yeast and mammalian sterol isomerases we propose that the sigma1-binding site is localized on a sterol isomerase related protein, involved in postsqualene sterol biosynthesis. PMID:9146879

  14. Cesium cation affinities and basicities

    NASA Astrophysics Data System (ADS)

    Gal, Jean-François; Maria, Pierre-Charles; Massi, Lionel; Mayeux, Charly; Burk, Peeter; Tammiku-Taul, Jaana

    2007-11-01

    This review focuses on the quantitative data related to cesium cation interaction with neutral or negatively charged ligands. The techniques used for measuring the cesium cation affinity (enthalpies, CCA), and cesium cation basicities (Gibbs free energies, CCB) are briefly described. The quantum chemical calculations methods that were specifically designed for the determination of cesium cation adduct structures and the energetic aspects of the interaction are discussed. The experimental results, obtained essentially from mass spectrometry techniques, and complemented by thermochemical data, are tabulated and commented. In particular, the correlations between cesium cation affinities and lithium cation affinities for the various kinds of ligands (rare gases, polyatomic neutral molecules, among them aromatic compounds and negative ions) serve as a basis for the interpretation of the diverse electrostatic modes of interaction. A brief account of some recent analytical applications of ion/molecule reactions with Cs+, as well as other cationization approaches by Cs+, is given.

  15. "Clickable" agarose for affinity chromatography.

    PubMed

    Punna, Sreenivas; Kaltgrad, Eiton; Finn, M G

    2005-01-01

    Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

  16. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  17. A multiplexed three-dimensional paper-based electrochemical impedance device for simultaneous label-free affinity sensing of total and glycated haemoglobin: The potential of using a specific single-frequency value for analysis.

    PubMed

    Boonyasit, Yuwadee; Chailapakul, Orawon; Laiwattanapaisal, Wanida

    2016-09-14

    A novel three-dimensional paper-based electrochemical impedance device (3D-PEID) is first introduced for measuring multiple diabetes markers. Herein, a simple 3D-PEID composed of a dual screen-printed electrode on wax-patterned paper coupled with a multilayer of magnetic paper was fabricated for label-free electrochemical detection. The results clearly demonstrated in a step-wise manner that the haptoglobin (Hp)-modified and 3-aminophenylboronic acid (APBA)-modified eggshell membranes (ESMs) were highly responsive to a clinically relevant range of total (0.5-20 g dL(-1); r(2) = 0.989) and glycated haemoglobin (HbA1c) (2.3%-14%; r(2) = 0.997) levels with detection limits (S/N = 3) of 0.08 g dL(-1) and 0.21%, respectively. The optimal binding frequencies of total haemoglobin and HbA1c to their specific recognition elements were 5.18 Hz and 9.99 Hz, respectively. The within-run coefficients of variation (CV) were 1.84%, 2.18%, 1.72%, and 2.01%, whereas the run-to-run CVs were 2.11%, 2.41%, 2.08%, and 2.21%, when assaying two levels of haemoglobin and HbA1c, respectively. The CVs for the haemoglobin and HbA1c levels measured on ten independently fabricated paper-based sheets were 1.96% and 2.10%, respectively. These results demonstrated that our proposed system achieved excellent precision for the simultaneous detection of total haemoglobin and HbA1c, with an acceptable reproducibility of fabrication. The long-term stability of the Hp-modified eggshell membrane (ESM) was 98.84% over a shelf-life of 4 weeks, enabling the possibility of storage or long-distance transport to remote regions, particularly in resource-limited settings; however, for the APBA-modified ESM, the stability was 92.35% over a one-week period. Compared with the commercial automated method, the results demonstrated excellent agreement between the techniques (p-value < 0.05), thus permitting the potential application of 3D-PEID for the monitoring of the glycaemic status in diabetic

  18. A multiplexed three-dimensional paper-based electrochemical impedance device for simultaneous label-free affinity sensing of total and glycated haemoglobin: The potential of using a specific single-frequency value for analysis.

    PubMed

    Boonyasit, Yuwadee; Chailapakul, Orawon; Laiwattanapaisal, Wanida

    2016-09-14

    A novel three-dimensional paper-based electrochemical impedance device (3D-PEID) is first introduced for measuring multiple diabetes markers. Herein, a simple 3D-PEID composed of a dual screen-printed electrode on wax-patterned paper coupled with a multilayer of magnetic paper was fabricated for label-free electrochemical detection. The results clearly demonstrated in a step-wise manner that the haptoglobin (Hp)-modified and 3-aminophenylboronic acid (APBA)-modified eggshell membranes (ESMs) were highly responsive to a clinically relevant range of total (0.5-20 g dL(-1); r(2) = 0.989) and glycated haemoglobin (HbA1c) (2.3%-14%; r(2) = 0.997) levels with detection limits (S/N = 3) of 0.08 g dL(-1) and 0.21%, respectively. The optimal binding frequencies of total haemoglobin and HbA1c to their specific recognition elements were 5.18 Hz and 9.99 Hz, respectively. The within-run coefficients of variation (CV) were 1.84%, 2.18%, 1.72%, and 2.01%, whereas the run-to-run CVs were 2.11%, 2.41%, 2.08%, and 2.21%, when assaying two levels of haemoglobin and HbA1c, respectively. The CVs for the haemoglobin and HbA1c levels measured on ten independently fabricated paper-based sheets were 1.96% and 2.10%, respectively. These results demonstrated that our proposed system achieved excellent precision for the simultaneous detection of total haemoglobin and HbA1c, with an acceptable reproducibility of fabrication. The long-term stability of the Hp-modified eggshell membrane (ESM) was 98.84% over a shelf-life of 4 weeks, enabling the possibility of storage or long-distance transport to remote regions, particularly in resource-limited settings; however, for the APBA-modified ESM, the stability was 92.35% over a one-week period. Compared with the commercial automated method, the results demonstrated excellent agreement between the techniques (p-value < 0.05), thus permitting the potential application of 3D-PEID for the monitoring of the glycaemic status in diabetic

  19. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  20. Making Bullying Prevention a Priority in Finnish Schools: The KiVa Antibullying Program

    ERIC Educational Resources Information Center

    Salmivalli, Christina; Poskiparta, Elisa

    2012-01-01

    The KiVa antibullying program has been widely implemented in Finnish comprehensive schools since 2009. The program is predicated on the idea that a positive change in the behaviors of classmates can reduce the rewards gained by the perpetrators of bullying and consequently their motivation to bully in the first place. KiVa involves both universal…

  1. Registration of the Ki14 × B73 recombinant inbred mapping population of maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Ohio Agricultural Research and Development Center released Ki14 × B73 (KB) maize (Zea mays L.) mapping population, a set of 119 recombinant inbred lines (RILs), in March 2007. The mapping population was derived from a biparental cross between inbreds Ki14 (NCRPIS accession Ames 27259) and B73 (...

  2. Selective and high affinity labeling of neuronal and recombinant nociceptin receptors with the hexapeptide radioprobe [(3)H]Ac-RYYRIK-ol.

    PubMed

    Bojnik, Engin; Farkas, Judit; Magyar, Anna; Tömböly, Csaba; Güçlü, Umit; Gündüz, Ozge; Borsodi, Anna; Corbani, Maïthe; Benyhe, Sándor

    2009-12-01

    The synthetic hexapeptide Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) represents a highly potent and selective partial agonist ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (nociceptin receptor, NOPr). Ac-RYYRIK-ol has been labeled with tritium yielding [(3)H]Ac-RYYRIK-ol with exceptionally high specific radioactivity of 94Ci/mmol. The radioprobe is chemically stable even at 24 degrees C in ethanol solution for at least 4 days. No significant decomposition of the [(3)H]ligand occurred under the condition of the binding experiments indicating a fine enzymatic stability of the peptide. Radioreceptor binding studies were conducted using native neuronal NOPr preparation of rat brain membrane fractions and recombinant human nociceptin receptor ((h)NOPr) preparations from cultured Chinese Hamster Ovary (CHO) cells stably expressing (h)NOPr. Specific binding of the compound was reversible, saturable and of high affinity. No cross-reaction with the opioid receptors was observed suggesting superior NOPr selectivity of the ligand. Monophasic isotherm curves obtained in radioligand binding saturation and homologous displacement experiments indicated the presence of single binding sites in both preparations. Average densities of the [(3)H]Ac-RYYRIK-ol recognition sites were 237 and 749fmol/mg protein in rat brain and transfected cells, respectively. Equilibrium affinity values (K(d)s) were determined by three independent way providing identical results. In rat brain membranes K(d)s of 0.3-1.3nM were found depending upon the assay type. In homologous competition studies performed on (h)NOP-CHO cell membranes almost the same binding affinities were measured for Ac-RYYRIK-ol either with [(3)H]Ac-RYYRIK-ol (K(i) 2.8nM) or with [(3)H](Leu(14))nociceptin (2.3nM). A number of NOPr and opioid ligands were screened in heterologous displacement experiments and displayed a rank order of affinity profile being consistent with fairly good NOPr selectivity of the sites

  3. Bodilisant—A Novel Fluorescent, Highly Affine Histamine H3 Receptor Ligand

    PubMed Central

    2012-01-01

    A piperidine-based lead structure for the human histamine H3 receptor (hH3R) was coupled with the BODIPY fluorophore and resulted in a strong green fluorescent (quantum yield, 0.92) hH3R ligand with affinity in the nanomolar concentration range (Ki hH3R = 6.51 ± 3.31 nM), named Bodilisant. Screening for affinities at histamine and dopamine receptor subtypes showed high hH3R preference. Bodilisant was used for visualization of hH3R in hH3R overexpressing HEK-293 cells with fluorescence confocal laser scanning microscopy. In addition, in native human brain tissues, Bodilisant showed clear and displaceable images of labeled hH3R. PMID:24900647

  4. Effectors of hemoglobin. Separation of allosteric and affinity factors.

    PubMed Central

    Marden, M C; Bohn, B; Kister, J; Poyart, C

    1990-01-01

    The relative contributions of the allosteric and affinity factors toward the change in p50 have been calculated for a series of effectors of hemoglobin (Hb). Shifts in the ligand affinity of deoxy Hb and the values for 50% ligand saturation (p50) were obtained from oxygen equilibrium data. Because the high-affinity parameters (liganded conformation) are poorly determined from the equilibrium curves, they were determined from kinetic measurements of the association and dissociation rates with CO as ligand. The CO on-rates were obtained by flash photolysis measurements. The off-rates were determined from the rate of oxidation of HbCO by ferricyanide, or by replacement of CO with NO. The partition function of fully liganded hemoglobin for oxygen and CO is only slightly changed by the effectors. Measurements were made in the presence of the effectors 2,3-diphosphoglycerate (DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), and two recently synthesized derivatives of Bzf (LR16 and L35). Values of p50 change by over a factor of 60; the on-rates decrease by nearly a factor of 8, with little change in the off-rates for the liganded conformation. The data indicate that both allosteric and affinity parameters are changed by the effectors; the changes in ligand affinity represent the larger contribution toward shifts in p50. PMID:2306490

  5. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  6. Ki-67 index and response to chemotherapy in patients with neuroendocrine tumours.

    PubMed

    Childs, Alexa; Kirkwood, Amy; Edeline, Julien; Luong, Tu Vinh; Watkins, Jennifer; Lamarca, Angela; Alrifai, Doraid; Nsiah-Sarbeng, Phyllis; Gillmore, Roopinder; Mayer, Astrid; Thirlwell, Christina; Sarker, Debashis; Valle, Juan W; Meyer, Tim

    2016-07-01

    Chemotherapy (CT) is widely used for neuroendocrine tumours (NETs), but there are no validated biomarkers to predict response. The Ki-67 proliferation index has been proposed as a means of selecting patients for CT, but robust data are lacking. The aim of this study was to investigate the relationship between response to chemotherapy and Ki-67 in NET. We reviewed data from 222 NET patients treated with CT. Tumours were graded according to Ki-67 index: G1 ≤2%, G2 3-20% and G3 >20%. Response was assessed according to RECIST and survival calculated from start of chemotherapy to death. To explore Ki-67 as a marker of response, we calculated the likelihood ratio and performed receiver operating characteristic analysis. Overall, 193 patients had a documented Ki-67 index, of which 173 were also evaluable for radiological response: 10% were G1, 46% G2 and 43% G3; 46% were pancreatic NET (PNET). Median overall survival was 22.1 months. Overall response rate was 30% (39% in PNET vs 22% in non-PNET) and 43% of patients had stable disease. Response rate increased with grade: 6% in G1 tumours, 24% in G2 and 43% in G3. However, maximum likelihood ratio was 2.3 at Ki-67=35%, and the area under the ROC curve was 0.60. As reported previously, a high Ki-67 was an adverse prognostic factor for overall survival. In conclusion, response to CT increases with Ki-67 index, but Ki-67 alone is an unreliable means to select patients for CT. Improved methods to stratify patients for systemic therapy are required. PMID:27412968

  7. Ki-67 immunostaining in astrocytomas: Association with histopathological grade – A South Indian study

    PubMed Central

    Shivaprasad, Nandish Vastrad; Satish, Suchitha; Ravishankar, Sunila; Vimalambike, Manjunath Gubbi

    2016-01-01

    Background: Astrocytomas are the most common primary tumor of the central nervous system. The distinction between different tumor grades can be tested despite criteria given by the World Health Organization (WHO). Ki-67 is a potent biological marker used in grading of astrocytomas, which estimates growth of the neoplasm quantitatively and will help in predicting prognosis accurately. Objectives: The aim of this was to study the proliferative activity using Ki-67 immunostaining and to assess the relationship of Ki-67 staining with the histopathological grading of astrocytomas. Patients and Methods: Thirty cases of histologically proven astrocytomas were studied. The histopathological grade was assessed using the 2007 WHO criteria. Immunohistochemistry for Ki-67 was done on paraffin-embedded wax sections. P < 0.05 was considered statistically significant. Results: Thirty cases of astrocytomas studied showed a male preponderance (M:F = 1.72:1) with a mean age of 48.1 years. Of these, Grade I, (n = 1, 3.33%), Grade II, (n = 7, 23.3%), Grade III (n = 6, 20%), and Grade IV (n = 16, 53.3%) astrocytomas were analyzed. The mean Ki-67 labeling index (LI) in Grades I, II, III, and IV was 0.02, 0.81, 9.14, and 17.81, respectively. Statistically significant difference was seen in the Ki-67 LI of low-grade (Grade II) and high-grade astrocytomas (Grades III and IV). There was concordance between histopathological grading and Ki-67 LI in 27 (90%) and discordance in 3 (10%) cases. Conclusion: Ki-67 LI varies considerably in different grades of astrocytomas and considerable overlaps can be observed between them. It can be of great help in situations where there is a lack of correlation between clinical parameters and histopathological diagnosis. Determination of Ki-67 LI should constitute a part of routine investigations in patients with astrocytomas.

  8. Expression of Ki-67 in normal oral epithelium, leukoplakic oral epithelium and oral squamous cell carcinoma

    PubMed Central

    Birajdar, Smita Shrishail; Radhika, MB; Paremala, K; Sudhakara, M; Soumya, M; Gadivan, Mohsin

    2014-01-01

    Aims and Objective: To demonstrate the presence, location and pattern of cell proliferation in different histological grades of oral epithelial dysplasia (OED), oral squamous cell carcinoma (OSCC) and normal oral epithelium (NOE) using an antibody directed against the Ki-67 antigen and its intensity of staining evaluated respectively. Materials and Methods: A total number of 100 archival paraffin embedded blocks obtained from Department of Oral and Maxillofacial Pathology were studied. The case details were retrieved which consisted of histopathologically diagnosed cases of OSCC (n = 20), low risk OED (n = 30), high risk OED (n = 30) and normal appearing mucosa (n = 20) were taken as standard for comparison. Ki-67 immunostaining was detected. Ki-67 positive cells were counted in the five random high power fields in each case. Results: Ki-67 labeling Index (LI) was restricted to the basal and parabasal layers of the normal oral epithelium irrespective of age, sex and site whereas it was seen in the basal, suprabasal and spinous layers in OED. Ki-67 LI is increased in high risk cases than the low risk cases of OED. Ki-67 positive cells in OSCC were located in the periphery of the tumor nests than the center, where frequent mitoses were observed. Conclusion: The architectural alteration evaluated by Ki-67 antibody in proliferating cell distribution in the layers of epithelial dysplasias may provide useful information to evaluate the grading of OED. Ki-67 LI increased in high risk cases than low risk cases of OED. This study showed that over expression of Ki-67 antigen between well-differentiated and poorly differentiated OSCC was in accordance with histologic grade of malignancy but not in accordance with moderately differentiated OSCC. PMID:25328294

  9. Ki-67 immunostaining in astrocytomas: Association with histopathological grade – A South Indian study

    PubMed Central

    Shivaprasad, Nandish Vastrad; Satish, Suchitha; Ravishankar, Sunila; Vimalambike, Manjunath Gubbi

    2016-01-01

    Background: Astrocytomas are the most common primary tumor of the central nervous system. The distinction between different tumor grades can be tested despite criteria given by the World Health Organization (WHO). Ki-67 is a potent biological marker used in grading of astrocytomas, which estimates growth of the neoplasm quantitatively and will help in predicting prognosis accurately. Objectives: The aim of this was to study the proliferative activity using Ki-67 immunostaining and to assess the relationship of Ki-67 staining with the histopathological grading of astrocytomas. Patients and Methods: Thirty cases of histologically proven astrocytomas were studied. The histopathological grade was assessed using the 2007 WHO criteria. Immunohistochemistry for Ki-67 was done on paraffin-embedded wax sections. P < 0.05 was considered statistically significant. Results: Thirty cases of astrocytomas studied showed a male preponderance (M:F = 1.72:1) with a mean age of 48.1 years. Of these, Grade I, (n = 1, 3.33%), Grade II, (n = 7, 23.3%), Grade III (n = 6, 20%), and Grade IV (n = 16, 53.3%) astrocytomas were analyzed. The mean Ki-67 labeling index (LI) in Grades I, II, III, and IV was 0.02, 0.81, 9.14, and 17.81, respectively. Statistically significant difference was seen in the Ki-67 LI of low-grade (Grade II) and high-grade astrocytomas (Grades III and IV). There was concordance between histopathological grading and Ki-67 LI in 27 (90%) and discordance in 3 (10%) cases. Conclusion: Ki-67 LI varies considerably in different grades of astrocytomas and considerable overlaps can be observed between them. It can be of great help in situations where there is a lack of correlation between clinical parameters and histopathological diagnosis. Determination of Ki-67 LI should constitute a part of routine investigations in patients with astrocytomas. PMID:27695229

  10. Odorant-binding proteins display high affinities for behavioral attractants and repellents in the natural predator Chrysopa pallens.

    PubMed

    Li, Zhao-Qun; Zhang, Shuai; Luo, Jun-Yu; Wang, Si-Bao; Dong, Shuang-Lin; Cui, Jin-Jie

    2015-07-01

    Chrysopa pallens is an important natural predator of various pests in many different cropping systems. Understanding the sophisticated olfactory system of insect antennae is crucial for studying the physiological bases of olfaction and could also help enhance the effectiveness of C. pallens in biological control. However, functional studies of the olfactory genes in C. pallens are still lacking. In this study, we cloned five odorant-binding protein (OBP) genes from C. pallens (CpalOBPs). Quantitative RT-PCR results indicated that the five CpalOBPs had different tissue expression profiles. Ligand-binding assays showed that farnesol, farnesene, cis-3-hexenyl hexanoate, geranylacetone, beta-ionone, octyl aldehyde, decanal, nerolidol (Ki<20 μM), and especially 2-pentadecanone (Ki=1.19 μM) and 2-hexyl-1-decanol (Ki=0.37 μM) strongly bound to CpalOBP2. CpalOBP15 exhibited high binding affinities for beta-ionone, 2-tridecanone, trans-nerolidol, and dodecyl aldehyde. Behavioral trials using the 14 compounds exhibiting high binding affinities for the CpalOBPs revealed that nine were able to elicit significant behavioral responses from C. pallens. Among them, farnesene and its corresponding alcohol, farnesol, elicited remarkable repellent behavioral responses from C. pallens. Our study provides several compounds that could be selected to develop slow-release agents that attract/repel C. pallens and to improve the search for strategies to eliminate insect pests. PMID:25810363

  11. Synthesis and structure-activity relationship studies of novel 2-diarylethyl substituted (2-carboxycycloprop-1-yl)glycines as high-affinity group II metabotropic glutamate receptor ligands.

    PubMed

    Sørensen, Ulrik S; Bleisch, Thomas J; Kingston, Anne E; Wright, Rebecca A; Johnson, Bryan G; Schoepp, Darryle D; Ornstein, Paul L

    2003-01-17

    The major excitatory neurotransmitter in the central nervous system, (S)-glutamic acid , activates both ionotropic and metabotropic excitatory amino acid receptors. Its importance in connection to neurological and psychiatric disorders has directed great attention to the development of compounds that modulate the effects of this endogenous ligand. Whereas L-carboxycyclopropylglycine (L-CCG-1) is a potent agonist at, primarily, group II metabotropic glutamate receptors, alkylation of at the alpha-carbon notoriously result in group II mGluR antagonists, of which the most potent compound described so far, LY341495, displays IC(50) values of 23 and 10 nM at the group II receptor subtypes mGlu2 and mGlu3, respectively. In this study we synthesized a series of structural analogues of in which the xanthyl moiety is replaced by two substituted-phenyl groups. The pharmacological characterization shows that these novel compounds have very high affinity for group II mGluRs when tested as their racemates. The most potent analogues demonstrate K(i) values in the range of 5-12 nM, being thus comparable to LY341495. PMID:12470714

  12. Quantification of hydrophobic interaction affinity of colloids

    NASA Astrophysics Data System (ADS)

    Saini, G.; Nasholm, N.; Wood, B. D.

    2009-12-01

    Colloids play an important role in a wide variety of disciplines, including water and wastewater treatment, subsurface transport of metals and organic contaminants, migration of fines in oil reservoirs, biocolloid (virus and bacteria) transport in subsurface, and are integral to laboratory transport studies. Although the role of hydrophobicity in adhesion and transport of colloids, particularly bacteria, is well known; there is scarcity of literature regarding hydrophobicity measurement of non-bacterial colloids and other micron-sized particles. Here we detail an experimental approach based on differential partitioning of colloids between two liquid phases (hydrocarbon and buffer) as a measure of the hydrophobic interaction affinity of colloids. This assay, known as Microbial adhesion to hydrocarbons or MATH, is frequently used in microbiology and bacteriology for quantifying the hydrophobicity of microbes. Monodispersed colloids and particles, with sizes ranging from 1 micron to 33 micron, were used for the experiments. A range of hydrophobicity values were observed for different particles. The hydrophobicity results are also verified against water contact angle measurements of these particles. This liquid-liquid partitioning assay is quick, easy-to-perform and requires minimal instrumentation. Estimation of the hydrophobic interaction affinity of colloids would lead to a better understanding of their adhesion to different surfaces and subsequent transport in porous media.

  13. A Sustainable and Efficient Synthesis of Benzyl Phosphonates Using PEG/KI Catalytic System.

    PubMed

    Disale, Shamrao; Kale, Sandip; Abraham, George; Kahandal, Sandeep; Sawarkar, Ashish N; Gawande, Manoj B

    2016-01-01

    An efficient and expedient protocol for the synthesis of benzyl phosphonates using KI/K2CO3 as a catalytic system and PEG-400 as benign solvent has been developed. The reaction proceeds smoothly at room temperature achieving excellent selectivity and yield of the corresponding products. The combination of PEG-400, KI, and K2CO3 in this reaction avoids the need of volatile/toxic organic solvents and reactive alkali metals or metal nanoparticles/hydrides. We believe that this benign combination (PEG-400 and KI) could be used for other related organic transformations. PMID:27579301

  14. A Sustainable and Efficient Synthesis of Benzyl Phosphonates Using PEG/KI Catalytic System

    PubMed Central

    Disale, Shamrao; Kale, Sandip; Abraham, George; Kahandal, Sandeep; Sawarkar, Ashish N.; Gawande, Manoj B.

    2016-01-01

    An efficient and expedient protocol for the synthesis of benzyl phosphonates using KI/K2CO3 as a catalytic system and PEG-400 as benign solvent has been developed. The reaction proceeds smoothly at room temperature achieving excellent selectivity and yield of the corresponding products. The combination of PEG-400, KI, and K2CO3 in this reaction avoids the need of volatile/toxic organic solvents and reactive alkali metals or metal nanoparticles/hydrides. We believe that this benign combination (PEG-400 and KI) could be used for other related organic transformations. PMID:27579301

  15. A sustainable and efficient synthesis of benzyl phosphonates using PEG/KI catalytic system

    NASA Astrophysics Data System (ADS)

    Gawande, Manoj; Disale, Shamrao; Kale, Sandip; Abraham, George; Kahandal, Sandeep; Sawarkar, Ashish

    2016-08-01

    An efficient and expedient protocol for the synthesis of benzyl phosphonates using KI/K2CO3 as a catalytic system and PEG-400 as benign solvent has been developed. The reaction proceeds smoothly at room temperature achieving excellent selectivity and yield of the corresponding products. The combination of PEG-400, KI and K2CO3 in this reaction avoids the need of volatile/toxic organic solvents and reactive alkali metals or metal nanoparticles/hydrides. We believe that this benign combination (PEG-400 and KI) could be used for other related organic transformations.

  16. Enhancing ligand-protein binding in affinity thermoprecipitation: elucidation of spacer effects

    PubMed

    Vaidya; Lele; Kulkarni; Mashelkar

    1999-08-20

    Copolymers of N-isopropylacrylamide and N-acryloyl amino acid spacers of varying chain length were synthesized. p-Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (Ki) of these polymers for trypsin decreased, i. e., the efficiency of PABA-trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower Ki than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N-acyl-p-aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect. Copyright 1999 John Wiley & Sons, Inc. PMID:10397880

  17. Two-parameter twisted quantum affine algebras

    NASA Astrophysics Data System (ADS)

    Jing, Naihuan; Zhang, Honglian

    2016-09-01

    We establish Drinfeld realization for the two-parameter twisted quantum affine algebras using a new method. The Hopf algebra structure for Drinfeld generators is given for both untwisted and twisted two-parameter quantum affine algebras, which include the quantum affine algebras as special cases.

  18. Association of GATA3, P53, Ki67 status and vascular peritumoral invasion are strongly prognostic in luminal breast cancer

    PubMed Central

    Jacquemier, Jocelyne; Charafe-Jauffret, Emmanuelle; Monville, Florence; Esterni, Benjamin; Extra, Jean Marc; Houvenaeghel, Gilles; Xerri, Luc; Bertucci, François; Birnbaum, Daniel

    2009-01-01

    Introduction Breast cancers are traditionally divided into hormone-receptor positive and negative cases. This classification helps to guide patient management. However, a subgroup of hormone-receptor positive patients relapse irrespective of hormonal therapy. Gene expression profiling has classified breast tumours into five major subtypes with significant different outcome. The two luminal subtypes, A and B, show high expression of ESR1, GATA3 and FOXA1 genes. Prognostic biomarkers for oestrogen receptor (ER)-positive cases include progesterone receptor (PR) and androgen receptor (AR), and proteins related to proliferation or apoptotic resistance. The aim of this study was to identify the best predictors of success of hormonal therapy. Methods By immunohistochemistry we studied 10 markers in a consecutive series of 832 cases of breast carcinoma treated at the Paoli-Calmettes Institute from 1990 to 2002 and deposited onto tissue microarrays (TMA). These markers were luminal-related markers ER, PR, AR, FOXA1 and GATA3 transcription factors, proliferation-related Ki67 and CCND1, ERBB2, anti-apoptotic BCL2 and P53. We also measured vascular peritumoural invasion (VPI), size, grade and lymph node involvement. For 143 cases, gene expression profiles were available. Adjuvant chemotherapy and hormonal therapy were given to high- and low-risk patients, respectively. The 162 events observed and taken into account were metastases. Results Molecular expression of the 10 parameters and subtype with ER status were strongly correlated. Of the 67 luminal A cases of this series, 63 were ER-positive. Multivariate analyses showed the highly significant prognostic value of VPI (hazard ratio (HR) = 2.47), Ki67 (HR = 2.9), P53 (HR = 2.9) and GATA3 (HR = 0.5) for the 240 patients who received hormonal therapy. Conclusions A panel of three antibodies (Ki67, P53 and GATA3) associated with VPI can significantly improve the traditional prognosticators in predicting outcome for ER

  19. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

    NASA Astrophysics Data System (ADS)

    Imaduwage, Kasun P.; Go, Eden P.; Zhu, Zhikai; Desaire, Heather

    2016-09-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods.

  20. Design of High-Affinity Stapled Peptides To Target the Repressor Activator Protein 1 (RAP1)/Telomeric Repeat-Binding Factor 2 (TRF2) Protein-Protein Interaction in the Shelterin Complex.

    PubMed

    Ran, Xu; Liu, Liu; Yang, Chao-Yie; Lu, Jianfeng; Chen, Yong; Lei, Ming; Wang, Shaomeng

    2016-01-14

    Shelterin, a six-protein complex, plays a fundamental role in protecting both the length and the stability of telomeres. Repressor activator protein 1 (RAP1) and telomeric repeat-binding factor 2 (TRF2) are two subunits in shelterin that interact with each other. Small-molecule inhibitors that block the RAP1/TRF2 protein-protein interaction can disrupt the structure of shelterin and may be employed as pharmacological tools to investigate the biology of shelterin. On the basis of the cocrystal structure of RAP1/TRF2 complex, we have developed first-in-class triazole-stapled peptides that block the protein-protein interaction between RAP1 and TRF2. Our most potent stapled peptide binds to RAP1 protein with a Ki value of 7 nM and is >100 times more potent than the corresponding wild-type TRF2 peptide. On the basis of our high-affinity peptides, we have developed and optimized a competitive, fluorescence polarization (FP) assay for accurate and rapid determination of the binding affinities of our designed compounds and this assay may also assist in the discovery of non-peptide, small-molecule inhibitors capable of blocking the RAP1/TRF2 protein-protein interaction.

  1. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

    NASA Astrophysics Data System (ADS)

    Imaduwage, Kasun P.; Go, Eden P.; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods.

  2. N-(4-(4-(2,3-Dichloro- or 2-methoxyphenyl)piperazin-1-yl)-butyl)-heterobiarylcarboxamides with Functionalized Linking Chains as High Affinity and Enantioselective D3 Receptor Antagonistsγ

    PubMed Central

    Newman, Amy Hauck; Grundt, Peter; Cyriac, George; Deschamps, Jeffrey R.; Taylor, Michelle; Kumar, Rakesh; Ho, David; Luedtke, Robert R.

    2009-01-01

    In the present report, the D3 receptor pharmacophore is modified in the 2,3-diCl-and 2-OCH3-phenyl piperazine class of compounds with the goal to improve D3 receptor affinity and selectivity. This extension of structure-activity relationships (SAR) has resulted in the identification of the first enantioselective D3 antagonists (R- and S-22) to be reported, wherein enantioselectivity is more pronounced at D3 than at D2, and that a binding region on the second extracellular loop (E2) may play a role in both enantioselectivity and D3 receptor selectivity. Moreover, we have discovered some of the most D3-selective compounds reported to date that show high affinity (Ki =1 nM) for D3 and ∼400-fold selectivity over the D2 receptor subtype. Several of these analogues showed exquisite selectivity for D3 receptors over >60 other receptors further underscoring their value as in vivo research tools. These lead compounds also have appropriate physical characteristics for in vivo exploration and therefore will be useful in determining how intrinsic activity at D3 receptors tested in vitro is related to behaviors in animal models of addiction and other neuropsychiatric disorders. PMID:19331412

  3. Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

    PubMed Central

    Millward, Steven W.; Fiacco, Stephen; Austin, Ryan J.; Roberts, Richard W.

    2012-01-01

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein Gαi1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic Gαi binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity (Ki ≈ 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the βγ heterodimer, an endogenous Gαi1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces. PMID:17894440

  4. Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics.

    PubMed Central

    Wright, C L; Warsy, A S; Holroyde, M J; Trayer, I P

    1978-01-01

    The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity

  5. Ki-67 immunostaining in uveal melanoma. The effect of pre-enucleation radiotherapy

    SciTech Connect

    Mooy, C.M.; de Jong, P.T.; Van der Kwast, T.H.; Mulder, P.G.; Jager, M.J.; Ruiter, D.J. )

    1990-10-01

    The reactivity of 33 choroidal and ciliary body melanomas with monoclonal antibody Ki-67, which recognizes a proliferation associated nuclear antigen, has been assessed and compared with clinicopathologic parameters. In 23 cases, 8 Gy irradiation was given 2 days before enucleation. Nonirradiated melanomas had a significantly higher proliferation rate as defined by staining with monoclonal antibody Ki-67 as compared with irradiated tumors (P = 0.007). Similarly, a strong relationship was found between pre-enucleation irradiation and low mitotic activity (P = 0.001). There was no significant correlation between the presence of Ki-67-positive nuclei and histologic classification, largest tumor diameter, localization of the tumor, age, sex, scleral invasion, pigmentation, and lymphocytic infiltration. The relevance of Ki-67 immunohistochemistry for the assessment of the life prognosis of patients with uveal melanoma has to be studied prospectively.

  6. [The effect of blood serum polyreactive immunoglobulins on serum antibody affinity determination].

    PubMed

    Bobrovnik, S A; Demchenko, M A; Komisarenko, S V

    2010-01-01

    The presence of polyreactive immunoglobulins in sera may substantially influence on the accuracy of antibody affinity determination. In order to obtain precise values of antibody affinity one should apply one of two following ways. First, one should block polyreactive immunoglobulins with high concentration of Twin 20 and high concentration of any antigen which does not interact with studying antibody. After this antibody affinity may be determined by traditional methods. Another way is the application of the method suggested by us earlier, which allow determining affinity of two antibodies in a mixture and the relation of their concentrations.

  7. Antisense treatment directed against mutated Ki-ras in human colorectal adenocarcinoma

    PubMed Central

    Andreyev, H; Ross, P; Cunningham, D; Clarke, P

    2001-01-01

    BACKGROUND—Kirsten ras (Ki-ras) mutations are common in gastrointestinal cancer and one codon 12 mutation, glycine to valine, is particularly aggressive in colorectal cancer.
AIMS—To investigate if this valine point mutation could be targeted with antisense oligonucleotides and to determine the efficacy of any antisense/mRNA interaction.
METHODS—Twenty nine antisense oligonucleotides were screened against target and control Ki-ras RNA in a cell free system and against target and control cell lines in culture.
RESULTS—The activity and specificity of the oligonucleotides varied. Results for the individual oligonucleotides were consistent in a cell free model and in cell culture using two different uptake promoters. Only one oligonucleotide was specific in its cleavage of target Ki-ras mRNA in the cell free system and appeared specific in cell culture, although changes in Ki-ras mRNA and protein expression following a single treatment could not be detected. Experiments in the cell free system showed that the point mutation is relatively inaccessible to oligonucleotides. Other sites on the Ki-ras RNA molecule, away from the point mutation, can be targeted more effectively.
CONCLUSIONS—Successful targeting of the clinically relevant Ki-ras point mutation with antisense oligonucleotides is difficult because of RNA structure at the mutated site and is inefficient compared with other sites on the Ki-ras mRNA.


Keywords: Ki-ras mutation; antisense treatment; colorectal carcinoma PMID:11156646

  8. The Er/Ki-67 Proportion in Breast Tumours - An Immunohistochemical Study

    PubMed Central

    Rai, M K

    2016-01-01

    Introduction Breast tumours are classified as benign, proliferative and invasive tumours. Estrogen hormone influences the proliferative activity and progression of the tumour. Estrogen Receptor (ER) status and proliferative index (Ki 67) are important histopathological factors in the development and prognosis of these tumours. Aim The present study was aimed to evaluate the variations in ER and Ki-67 expression in three broad categories of breast lesions namely benign breast disease, proliferative breast disease and malignant breast disease. Materials and Methods ER% and Ki-67% was evaluated on the histopathological tissues of 15 patients each of benign, proliferative and invasive breast tumours. The ER+/ Ki-67± ratio was calculated and the variation of expression between the three categories was analyzed using student’s t-test. Pearson’s coefficient of correlation was used to correlate ER and Ki-67 positivity within each category. Results The mean ER+/Ki-67+ in benign, proliferative and invasive tumours was 0.81, 0.87 and 1.42 respectively. A statistically significant difference in ER+/Ki-67+ proportions was observed between proliferative breast disease category and malignant breast disease category and also between benign breast disease category and malignant breast disease category (p<0.05). However, no significant difference was observed in benign breast disease category and proliferative breast disease category (p>0.05). A significant correlation was observed in proliferative breast disease and malignant breast disease categories. However, no significant correlation was observed in benign breast disease category Conclusion ER+/Ki-67+ ratio is an important determinant of the invasive breast cancer and can be used to differentiate invasive cancers from benign and proliferative breast tumours. PMID:27190810

  9. Comparative quantitative study of Ki-67 antibody staining in 78 B and T cell malignant lymphoma (ML) using two image analyser systems.

    PubMed

    Caulet, S; Lesty, C; Raphael, M; Schoevaert, D; Brousset, P; Binet, J L; Diebold, J; Delsol, G

    1992-06-01

    Total Ki-67 stained area percentage was studied in 32 B and 46 T malignant lymphomas (ML) using two different image analyser systems (TAS, Leitz; SAMBA TM 2005, TITN) respectively. The total Ki-67 area percentage was highly correlated to the number of Ki-67 positive cellular profiles (B-ML, r = 0.93; T-ML, r = 0.88), indicating that area percentage is a reliable alternative method to the manual cell counting. Image analysis allows quicker measurements, appropriate to large and strictly lymphomatous regions. The cell image processor (SAMBA TM 2005, TITN) linked to a color video camera was more suitable for immunohistochemical sections and allowed more automated and faster measurements than the texture analyser (TAS, Leitz) linked with a black and white camera. Alkaline phosphatase technique with fast red as chromogen was more suitable for the detection of Ki-67 stained area by thresholding than peroxidase technique with aminoethylcarbazol or with diaminobenzidine as chromogens. Significant differences were found between low and high grade in B and T ML according to the Kiel classification (mean values +/- SD of 7.7 +/- 3.8% and 16.6 +/- 6.2% in B-ML and of 10.2 +/- 7.9% and 25.6 +/- 16.3% in T-ML respectively). In follicular B-ML, considering follicular areas only, values were comparable to high grade ML; angioimmunoblastic-lymphadenopathy-like (AILD-type) T-ML belonging to low grade ML showed similar values to pleomorphic T-ML with medium and/or large cells belonging to high grade ML.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1409077

  10. Ki-67 cytological index can distinguish well-differentiated from poorly differentiated pancreatic neuroendocrine tumors: a comparative cytohistological study of 53 cases.

    PubMed

    Carlinfante, Gabriele; Baccarini, Paola; Berretti, Debora; Cassetti, Tiziana; Cavina, Maurizio; Conigliaro, Rita; De Pellegrin, Alessandro; Di Tommaso, Luca; Fabbri, Carlo; Fornelli, Adele; Frasoldati, Andrea; Gardini, Giorgio; Losi, Luisa; Maccio, Livia; Manta, Raffaele; Pagano, Nico; Sassatelli, Romano; Serra, Silvia; Camellini, Lorenzo

    2014-07-01

    The Ki-67 labeling index has been found to bear prognostic significance in gastrointestinal neuroendocrine tumors (NETs), and it was recently incorporated in NET histological grading. Nevertheless, a reliable preoperative determination of NET grading could be useful in clinical practice. The aim of this study is to compare the results of Ki-67 labeling index, as measured on cytological samples and on surgical specimens of patients with pancreatic NETs (P-NETs). We also investigated whether concordance might be improved, using a 5 % (instead of 2 %) cutoff value for defining G2 tumors. We retrospectively identified 48 consecutive patients with 53 P-NETs, from our five institutions, and we measured Ki-67 labeling index on their cytological samples and surgical specimens. The traditional 2 % and the alternative 5 % cutoff values were used to classify G2 tumors. The concordance rate between cytological and histological grading was 46/53 (86.8 %; weighted κ statistic 0.77; 95 % confidence interval (95 % CI) 0.60-0.94). No cases of cytological G1-G2 NETs were upgraded to G3 neuroendocrine carcinoma (NEC) at histological grading. Cytology was found to be highly specific in the diagnosis of both G2 (94.1 %; 95 % CI 80.3-99.3) and G3 tumors (100.0 %; 95 % CI 92.8-100), but the sensitivity was poor for G2 NETs (66.7 %; 95 % CI 38.4-88.2) and high for the prediction of G3 NECs (100 %; 95 % CI 39.8-100.0). When the 5 % cutoff value was adopted, concordance rate was 49/53 (92.4 %; weighted κ 0.82; 95 % CI 0.64-1.00). In conclusion, Ki-67 cytological expression can distinguish well-differentiated (both G1 and G2) from poorly differentiated P-NETs, and it may be useful for their preoperative classification. PMID:24807732

  11. The importance of tissue handling of surgically removed breast cancer for an accurate assessment of the Ki-67 index

    PubMed Central

    Arima, Nobuyuki; Nishimura, Reiki; Osako, Tomofumi; Nishiyama, Yasuyuki; Fujisue, Mamiko; Okumura, Yasuhiro; Nakano, Masahiro; Tashima, Rumiko; Toyozumi, Yasuo

    2016-01-01

    Aim Insufficient attention for the Ki-67 immunohistochemistry has been given to the importance of tissue handling for surgical breast cancer specimens. We sought to investigate the effect of fixation status on the Ki-67. Methods We examined the effect of fixative, time to and duration of fixation using surgical specimens, and finally, compared the paired Ki-67 index in the tumour between core needle and surgical specimen. Results The Ki-67 was significantly higher when 10% neutral buffered formalin was used (p=0.0276). Insufficient fixation caused a drastic reduction in the Ki-67 index (p=0.0177), but not significant in oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Sixteen hours delayed time to fixation also caused a reduction of the Ki-67 (p=0.0284), but not significant in ER. Prolonged fixation significantly led to a gradual reduction in the Ki-67 in a time-dependent manner, but not in both ER and HER2. Finally, cutting the tumour before fixation improved fixation status and consequently caused an increased level of the Ki-67 index (p=0.0181), which resulted in a strong correlation of the Ki-67 between core needle and surgical specimen (r=0.8595). Conclusions Tissue handling of surgical specimen is critical for assessing the Ki-67 compared with ER and HER2. We should pay more attention to tissue fixation status for the standard assessment of the Ki-67 index. PMID:26420767

  12. Human macrophages can express the Hodgkin's cell-associated antigen Ki-1 (CD30).

    PubMed Central

    Andreesen, R.; Brugger, W.; Löhr, G. W.; Bross, K. J.

    1989-01-01

    The normal precursor of the neoplastic cell in Hodgkin's lymphoma is still unknown. Previous reports on the expression of the Hodgkin's cell-associated antigen Ki-1, CD30, on normal cells have been limited to activated lymphocytes. This study demonstrates, however, that cells of the macrophage lineage also are able to express the Ki-1 antigen. The Ki-1 antigen is absent from normal blood monocytes but expressed on up to 85% of macrophage-type cells developed during subsequent in vitro differentiation on Teflon membranes. Unlike other maturation-associated antigens, Ki-1 is found only at late stages of the macrophage primary cultures. Its expression can be enhanced by human interferon-gamma in a fashion similar to that of HLA-DR molecules. In addition, freshly explanted tumor cells from three patients with histopathologic and clinical features consistent with the diagnosis of true histiocytic lymphoma or malignant histiocytosis as well as the permanent cell line SU-DHL-1 could be demonstrated to express the Ki-1 antigen. The phenotype of histiocytic malignancy was further evaluated to be HLA-DR+MAX.26+CD25+-EMA+OKT9+Ki-1+. The results could indicate either that Hodgkin's lymphoma may arise not only from the lymphocyte but also from the macrophage lineage or may emphasize a macrophage involvement in the pathogenesis of this disease. Images Figure 3 PMID:2536522

  13. Evidence for the association of the human regulatory protein Ki-1/57 with the translational machinery.

    PubMed

    Gonçalves, Kaliandra de Almeida; Bressan, Gustavo Costa; Saito, Angela; Morello, Luis Gustavo; Zanchin, Nilson Ivo T; Kobarg, Jörg

    2011-08-19

    Ki-1/57 is a cytoplasmic and nuclear protein of 57 kDa first identified in malignant cells from Hodgkin's lymphoma. Based on yeast-two hybrid protein interaction we found out that Ki-1/57 interacts with adaptor protein RACK1 (receptor of activated kinase 1), CIRP (cold-inducible RNA-binding protein), RPL38 (ribosomal protein L38) and FXR1 (fragile X mental retardation-related protein 1). Since these proteins are involved in the regulation of translation we suspected that Ki-1/57 may have a role in it. We show by immunoprecipitation the association of Ki-1/57 with FMRP. Confocal microscopy revealed that Ki-1/57 colocalizes with FMRP/FXR1/2 to stress granules. Furthermore Ki-1/57 cosediments with free ribosomal particles and enhances translation, when tethered to a reporter mRNA, suggesting that Ki-1/57 may be involved in translational regulation.

  14. Fluorogenic Assay for Inhibitors of HIV-1 Protease with Sub-picomolar Affinity

    NASA Astrophysics Data System (ADS)

    Windsor, Ian W.; Raines, Ronald T.

    2015-08-01

    A fluorogenic substrate for HIV-1 protease was designed and used as the basis for a hypersensitive assay. The substrate exhibits a kcat of 7.4 s-1, KM of 15 μM, and an increase in fluorescence intensity of 104-fold upon cleavage, thus providing sensitivity that is unmatched in a continuous assay of HIV-1 protease. These properties enabled the enzyme concentration in an activity assay to be reduced to 25 pM, which is close to the Kd value of the protease dimer. By fitting inhibition data to Morrison’s equation, Ki values of amprenavir, darunavir, and tipranavir were determined to be 135, 10, and 82 pM, respectively. This assay, which is capable of measuring Ki values as low as 0.25 pM, is well-suited for characterizing the next generation of HIV-1 protease inhibitors.

  15. The region-of-interest size impacts on Ki67 quantification by computer-assisted image analysis in breast cancer.

    PubMed

    Christgen, Matthias; von Ahsen, Sabrina; Christgen, Henriette; Länger, Florian; Kreipe, Hans

    2015-09-01

    Therapeutic decision-making in breast cancer depends on histopathologic biomarkers and is influenced by the Ki67 proliferation index. Computer-assisted image analysis (CAIA) promises to improve Ki67 quantification. Several commercial applications have been developed for semiautomated CAIA-based Ki67 quantification, many of which rely on measurements in user-defined regions of interest (ROIs). Because of intratumoral proliferative heterogeneity, definition of the ROI is an important step in the analytical procedure. This study explores the ROI size impacts on Ki67 quantification. Whole-slide sections of 100 breast cancers were immunostained with the anti-Ki67 antibody 30-9 and were analyzed on the iScan Coreo digital pathology platform using a Food and Drug Administration-cleared Ki67 quantification software version v5.3 (Virtuoso; Ventana, Tucson, TX). For each case, the Ki67 labeling index (LI) was determined in multiple ROIs of gradually increasing size centered around a high-proliferation area. The spatial Ki67 decline was modeled with nonlinear regression. Depending on the ROI size, the median Ki67 LI varied between 55% and 15%. The proportion of tumors classified as Ki67 low according to the St Gallen 2013/2015 cutoff increased from 2% to 56%, as the ROI size increased from 50 to 10,000 cells captured. The interrater reliability of conventional Ki67 assessment versus CAIA-based Ki67 quantification was also dependent on the ROI size and varied between slight and almost perfect agreement (Cohen κ = 0.06-0.85). In conclusion, the ROI size is a critically important parameter for semiautomated Ki67 quantification by CAIA. Ki67 LIs determined on platforms like iScan Coreo/Virtuoso require an ROI size adjustment, for which we offer a downloadable data transformation tool.

  16. The region-of-interest size impacts on Ki67 quantification by computer-assisted image analysis in breast cancer.

    PubMed

    Christgen, Matthias; von Ahsen, Sabrina; Christgen, Henriette; Länger, Florian; Kreipe, Hans

    2015-09-01

    Therapeutic decision-making in breast cancer depends on histopathologic biomarkers and is influenced by the Ki67 proliferation index. Computer-assisted image analysis (CAIA) promises to improve Ki67 quantification. Several commercial applications have been developed for semiautomated CAIA-based Ki67 quantification, many of which rely on measurements in user-defined regions of interest (ROIs). Because of intratumoral proliferative heterogeneity, definition of the ROI is an important step in the analytical procedure. This study explores the ROI size impacts on Ki67 quantification. Whole-slide sections of 100 breast cancers were immunostained with the anti-Ki67 antibody 30-9 and were analyzed on the iScan Coreo digital pathology platform using a Food and Drug Administration-cleared Ki67 quantification software version v5.3 (Virtuoso; Ventana, Tucson, TX). For each case, the Ki67 labeling index (LI) was determined in multiple ROIs of gradually increasing size centered around a high-proliferation area. The spatial Ki67 decline was modeled with nonlinear regression. Depending on the ROI size, the median Ki67 LI varied between 55% and 15%. The proportion of tumors classified as Ki67 low according to the St Gallen 2013/2015 cutoff increased from 2% to 56%, as the ROI size increased from 50 to 10,000 cells captured. The interrater reliability of conventional Ki67 assessment versus CAIA-based Ki67 quantification was also dependent on the ROI size and varied between slight and almost perfect agreement (Cohen κ = 0.06-0.85). In conclusion, the ROI size is a critically important parameter for semiautomated Ki67 quantification by CAIA. Ki67 LIs determined on platforms like iScan Coreo/Virtuoso require an ROI size adjustment, for which we offer a downloadable data transformation tool. PMID:26206765

  17. Quantum dots-based double imaging combined with organic dye imaging to establish an automatic computerized method for cancer Ki67 measurement

    NASA Astrophysics Data System (ADS)

    Wang, Lin-Wei; Qu, Ai-Ping; Liu, Wen-Lou; Chen, Jia-Mei; Yuan, Jing-Ping; Wu, Han; Li, Yan; Liu, Juan

    2016-02-01

    As a widely used proliferative marker, Ki67 has important impacts on cancer prognosis, especially for breast cancer (BC). However, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study is to establish quantum dots (QDs)-based double imaging of nuclear Ki67 as red signal by QDs-655, cytoplasmic cytokeratin (CK) as yellow signal by QDs-585, and organic dye imaging of cell nucleus as blue signal by 4‧,6-diamidino-2-phenylindole (DAPI), and to develop a computer-aided automatic method for Ki67 index measurement. The newly developed automatic computerized Ki67 measurement could efficiently recognize and count Ki67-positive cancer cell nuclei with red signals and cancer cell nuclei with blue signals within cancer cell cytoplasmic with yellow signals. Comparisons of computerized Ki67 index, visual Ki67 index, and marked Ki67 index for 30 patients of 90 images with Ki67 ≤ 10% (low grade), 10% < Ki67 < 50% (moderate grade), and Ki67 ≥ 50% (high grade) showed computerized Ki67 counting is better than visual Ki67 counting, especially for Ki67 low and moderate grades. Based on QDs-based double imaging and organic dye imaging on BC tissues, this study successfully developed an automatic computerized Ki67 counting method to measure Ki67 index.

  18. Diagnostic performance of HPV E6/E7, hTERT, and Ki67 mRNA RT-qPCR assays on formalin-fixed paraffin-embedded cervical tissue specimens from women with cervical cancer.

    PubMed

    Wang, Hye-Young; Kim, Geehyuk; Cho, Hyemi; Kim, Sunghyun; Lee, Dongsup; Park, Sunyoung; Park, Kwang Hwa; Lee, Hyeyoung

    2015-06-01

    Human papillomavirus (HPV) is a major cause of cervical cancer, which is the third most common cancer in women. Human telomerase reverse transcriptase (hTERT) and Ki67 are tumor cell markers indicating cancer cell proliferation in cancer patients, and activation of hTERT and Ki67 leads to progressive cervical carcinogenesis. In the present study, we evaluated the CervicGen HPVE6/E7 mRNA RT-qDx assay, which detects 16 HPV high-risk (HR) genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 69), and the CervicGen hTERT and Ki67 mRNA RT-qDx assay using 117 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples. The diagnostic validity of the CervicGen HPV RT-qDx assay for detecting histologically proven prevalent squamous cell carcinoma (SCC) was 94% sensitivity, 100% specificity, 77.8% positive predictive value (PPV), and 78.9% negative predictive value (NPV). The most common HPV genotypes detected in FFPE cervical cancer tissue samples were HPV 16 (56%) and HPV 18 (10%). The positivity rate of hTERT and Ki67 mRNA expressions in FFPE cervical cancer tissue samples on RT-qPCR was 65% and 93% respectively. Moreover, the positivity rates were 92% for a combination of HPV E6/E7 and hTERT mRNA expressions, 97% for HPV E6/E7 and Ki67 mRNA expressions, and 99% (99/100) for the combination of HPV E6/E7, hTERT, and Ki67 mRNA expressions. These data showed that SSC FFPE cervical cancer tissue samples correlated more strongly with high Ki67 mRNA expressions than with hTERT mRNA expressions. Notably, hTERT and Ki67 mRNA expression level was increased in high-grade cervical lesions, but was very low in normal samples. Our findings suggest that the combination of HPV E6/E7, hTERT, and Ki67 mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions. Further studies are required to evaluate these assays as a useful predictive tool for screening low-grade cervical lesions.

  19. Antinociceptive action of DBO 17 and DBO 11 in mice: two 3,8 diazabicyclo (3.2.1.) octane derivates with selective mu opioid receptor affinity.

    PubMed

    Fadda, P; Barlocco, D; Tronci, S; Cignarella, G; Fratta, W

    1997-11-01

    Two 3,8 diazabicyclo (3.2.1.) octane derivates, namely DBO 17 and DBO 11, were studied for the opioid-like activity. In the rat brain membrane preparation binding studies, DBO 17 and DBO 11 showed a high affinity and selectivity for the mu opioid receptor (Ki's: 5.1 and 25 nM, respectively). DBO 17 and DBO 11 inhibited the nociceptive response in the hot-plate test of mice with ED50 values of 0.16 mg/kg and 0.44 mg/kg, respectively. The antinociceptive action of both DBO 17 and DBO 11 was blocked by naloxone. Tolerance to the antinociceptive action of DBO 17 and DBO 11 was present after 13 and 7 days of repeated treatment, respectively. Both DBO 17 and DBO 11 were ineffective in morphine-tolerant mice and vice versa. Chronic treatments (three times daily for seven consecutive days) of DBO 17 and DBO 11 induced a naloxone-precipitated withdrawal syndrome in DBO 17 treated mice similar to that in morphine treated mice, whereas in DBO 11 treated mice abstinence signs were virtually absent. These results indicate an interesting pharmacological profile that suggests these compounds as possible new candidates for the clinical treatment of pain.

  20. The maximal affinity of ligands

    PubMed Central

    Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

    1999-01-01

    We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin. PMID:10468550

  1. Engineering antibody affinity and specificity.

    PubMed

    Webster, D M; Roberts, S; Cheetham, J C; Griest, R; Rees, A R

    1988-01-01

    A combination of ab initio calculations, "knowledge-based prediction", molecular graphics and site-directed mutagenesis has enabled us to probe the molecular details of antibody:antigen recognition and binding and to alter the affinity and specificity of an antibody for its antigen. The significance of electrostatic hydrogen bonding, hydrophilic/hydrophobic patch matching and van der Waals interactions as well as CDR:CDR interactions are discussed in relation to the results of site-directed mutagenesis experiments on the anti-lysozyme antibody Gloop2. The ability to generate reconstructed antibodies, chimeric antibodies, catalytic antibodies and the use of modelled antibodies for the design of drugs is discussed. PMID:3209295

  2. Proton affinity of methyl nitrate - Less than proton affinity of nitric acid

    NASA Technical Reports Server (NTRS)

    Lee, Timothy J.; Rice, Julia E.

    1992-01-01

    Several state-of-the-art ab initio quantum mechanical methods were used to investigate the equilibrium structure, dipole moments, harmonic vibrational frequencies, and IR intensities of methyl nitrate, methanol, and several structures of protonated methyl nitrate, using the same theoretical methods as in an earlier study (Lee and Rice, 1992) of nitric acid. The ab initio results for methyl nitrate and methanol were found to be in good agreement with available experimental data. The proton affinity (PA) of methyl nitrate was calculated to be 176.9 +/-5 kcal/mol, in excellent agreement with the experimental value 176 kcal/mol obtained by Attina et al. (1987) and less than the PA value of nitric acid. An explanation of the discrepancy of the present results with those of an earlier study on protonated nitric acid is proposed.

  3. Dye affinity cryogels for plasmid DNA purification.

    PubMed

    Çimen, Duygu; Yılmaz, Fatma; Perçin, Işık; Türkmen, Deniz; Denizli, Adil

    2015-11-01

    The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA. PMID:26249596

  4. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, K.S.

    1996-04-01

    Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.

  5. Predicting Ki67% expression from DCE-MR images of breast tumors using textural kinetic features in tumor habitats

    NASA Astrophysics Data System (ADS)

    Chaudhury, Baishali; Zhou, Mu; Farhidzadeh, Hamidreza; Goldgof, Dmitry B.; Hall, Lawrence O.; Gatenby, Robert A.; Gillies, Robert J.; Weinfurtner, Robert J.; Drukteinis, Jennifer S.

    2016-03-01

    The use of Ki67% expression, a cell proliferation marker, as a predictive and prognostic factor has been widely studied in the literature. Yet its usefulness is limited due to inconsistent cut off scores for Ki67% expression, subjective differences in its assessment in various studies, and spatial variation in expression, which makes it difficult to reproduce as a reliable independent prognostic factor. Previous studies have shown that there are significant spatial variations in Ki67% expression, which may limit its clinical prognostic utility after core biopsy. These variations are most evident when examining the periphery of the tumor vs. the core. To date, prediction of Ki67% expression from quantitative image analysis of DCE-MRI is very limited. This work presents a novel computer aided diagnosis framework to use textural kinetics to (i) predict the ratio of periphery Ki67% expression to core Ki67% expression, and (ii) predict Ki67% expression from individual tumor habitats. The pilot cohort consists of T1 weighted fat saturated DCE-MR images from 17 patients. Support vector regression with a radial basis function was used for predicting the Ki67% expression and ratios. The initial results show that texture features from individual tumor habitats are more predictive of the Ki67% expression ratio and spatial Ki67% expression than features from the whole tumor. The Ki67% expression ratio could be predicted with a root mean square error (RMSE) of 1.67%. Quantitative image analysis of DCE-MRI using textural kinetic habitats, has the potential to be used as a non-invasive method for predicting Ki67 percentage and ratio, thus more accurately reporting high KI-67 expression for patient prognosis.

  6. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  7. Non-affine deformations in polymer hydrogels

    PubMed Central

    Wen, Qi; Basu, Anindita; Janmey, Paul A.; Yodh, A. G.

    2012-01-01

    Most theories of soft matter elasticity assume that the local strain in a sample after deformation is identical everywhere and equal to the macroscopic strain, or equivalently that the deformation is affine. We discuss the elasticity of hydrogels of crosslinked polymers with special attention to affine and non-affine theories of elasticity. Experimental procedures to measure non-affine deformations are also described. Entropic theories, which account for gel elasticity based on stretching out individual polymer chains, predict affine deformations. In contrast, simulations of network deformation that result in bending of the stiff constituent filaments generally predict non-affine behavior. Results from experiments show significant non-affine deformation in hydrogels even when they are formed by flexible polymers for which bending would appear to be negligible compared to stretching. However, this finding is not necessarily an experimental proof of the non-affine model for elasticity. We emphasize the insights gained from experiments using confocal rheoscope and show that, in addition to filament bending, sample micro-inhomogeneity can be a significant alternative source of non-affine deformation. PMID:23002395

  8. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  9. Affinity functions: recognizing essential parameters in fuzzy connectedness based image segmentation

    NASA Astrophysics Data System (ADS)

    Ciesielski, Krzysztof C.; Udupa, Jayaram K.

    2009-02-01

    Fuzzy connectedness (FC) constitutes an important class of image segmentation schemas. Although affinity functions represent the core aspect (main variability parameter) of FC algorithms, they have not been studied systematically in the literature. In this paper, we present a thorough study to fill this gap. Our analysis is based on the notion of equivalent affinities: if any two equivalent affinities are used in the same FC schema to produce two versions of the algorithm, then these algorithms are equivalent in the sense that they lead to identical segmentations. We give a complete characterization of the affinity equivalence and show that many natural definitions of affinity functions and their parameters used in the literature are redundant in the sense that different definitions and values of such parameters lead to equivalent affinities. We also show that two main affinity types - homogeneity based and object feature based - are equivalent, respectively, to the difference quotient of the intensity function and Rosenfeld's degree of connectivity. In addition, we demonstrate that any segmentation obtained via relative fuzzy connectedness (RFC) algorithm can be viewed as segmentation obtained via absolute fuzzy connectedness (AFC) algorithm with an automatic and adaptive threshold detection. We finish with an analysis of possible ways of combining different component affinities that result in non equivalent affinities.

  10. Immunoexpression of vascular endothelial growth factor and Ki-67 in human gingival samples: An observational study

    PubMed Central

    Kranti, K.; Mani, R.; Elizabeth, Anjana

    2015-01-01

    Aim: To evaluate immunohistochemically vascular endothelial growth factor (VEGF) and Ki-67 in human gingival samples and to compare these factors between healthy and diabetic patients. Materials and Methods: A total of 50 subjects were included in the study. They were categorized into three groups: Periodontally healthy group, periodontally diseased gingiva without any systemic disease group and periodontally diseased gingiva with controlled type II diabetes mellitus (DM) group. Gingival biopsies were performed and immunohistochemical analysis were done for VEGF and Ki-67 staining in gingival samples. Results: The present study found moderate intensity staining for VEGF in periodontitis group and periodontitis with controlled type II DM group and mild intensity staining for VEGF in periodontally healthy group. With regard to Ki-67, negative staining was observed in periodontally healthy group and mild staining in periodontitis group and periodontitis with controlled type II DM group. Conclusion: Further investigation needs to be conducted to identify how VEGF and Ki-67 are involved in the tissue inflammation associated processes and the relationship between VEGF and Ki-67 in progression of periodontitis. PMID:26097335

  11. The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism

    PubMed Central

    Kumar, Ganesan Senthil; Gokhan, Ezgi; De Munter, Sofie; Bollen, Mathieu; Vagnarelli, Paola; Peti, Wolfgang; Page, Rebecca

    2016-01-01

    Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes. DOI: http://dx.doi.org/10.7554/eLife.16539.001 PMID:27572260

  12. Apoptosis and Ki-67 as predictive factors for response to radiation therapy in feline nasal lymphomas

    PubMed Central

    FU, Dah-Renn; KATO, Daiki; ENDO, Yoshifumi; KADOSAWA, Tsuyoshi

    2016-01-01

    Nasal lymphoma is the most common nasal tumor in cats and is generally a solitary and radiosensitive tumor. We retrospectively evaluated the response to radiation and survival time in relation to apoptosis and Ki-67 indices in feline nasal lymphomas treated with radiation therapy. The apoptotic and Ki-67 indices were evaluated with TUNEL and immunohistochemical staining in 30 biopsy tissues that were taken before any treatment. These two indices were compared, and differences between different treatment response groups were analyzed. The correlation between the median survival times (MST) and the indices was estimated using the Kaplan Meier method, and statistical differences between survival curves were analyzed using a log-rank method. With regard to apoptotic index, a statistical difference was observed between the samples taken from cats with complete response and stable disease (1.22% vs. 0.45%; P=0.045). The Ki-67 index in cats with both complete response and partial response was significantly higher than in cats with stable disease (44.4% and 39.6% vs. 16.3%; P<0.001 and P=0.008, respectively). The cats with a high level of apoptosis (>0.9%) nasal lymphoma were not significantly prolonged MSTs (P=0.202), however, high Ki-67-positive (>40%) cats experienced a statistically significant relationship with longer survival time (P=0.015). Our results indicate that spontaneous apoptotic and Ki-67 indices are strong predictors for response to radiation therapy in feline nasal lymphomas. PMID:27086717

  13. The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism.

    PubMed

    Kumar, Ganesan Senthil; Gokhan, Ezgi; De Munter, Sofie; Bollen, Mathieu; Vagnarelli, Paola; Peti, Wolfgang; Page, Rebecca

    2016-01-01

    Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes. PMID:27572260

  14. Entropy based quantification of Ki-67 positive cell images and its evaluation by a reader study

    NASA Astrophysics Data System (ADS)

    Niazi, M. Khalid Khan; Pennell, Michael; Elkins, Camille; Hemminger, Jessica; Jin, Ming; Kirby, Sean; Kurt, Habibe; Miller, Barrie; Plocharczyk, Elizabeth; Roth, Rachel; Ziegler, Rebecca; Shana'ah, Arwa; Racke, Fred; Lozanski, Gerard; Gurcan, Metin N.

    2013-03-01

    Presence of Ki-67, a nuclear protein, is typically used to measure cell proliferation. The quantification of the Ki-67 proliferation index is performed visually by the pathologist; however, this is subject to inter- and intra-reader variability. Automated techniques utilizing digital image analysis by computers have emerged. The large variations in specimen preparation, staining, and imaging as well as true biological heterogeneity of tumor tissue often results in variable intensities in Ki-67 stained images. These variations affect the performance of currently developed methods. To optimize the segmentation of Ki-67 stained cells, one should define a data dependent transformation that will account for these color variations instead of defining a fixed linear transformation to separate different hues. To address these issues in images of tissue stained with Ki-67, we propose a methodology that exploits the intrinsic properties of CIE L∗a∗b∗ color space to translate this complex problem into an automatic entropy based thresholding problem. The developed method was evaluated through two reader studies with pathology residents and expert hematopathologists. Agreement between the proposed method and the expert pathologists was good (CCC = 0.80).

  15. Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease

    PubMed Central

    Bochtler, Matthias; Szczepanowski, Roman H; Tamulaitis, Gintautas; Grazulis, Saulius; Czapinska, Honorata; Manakova, Elena; Siksnys, Virginijus

    2006-01-01

    Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5′-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5′-overhangs. Here, we report the crystal structure of the Ecl18kI–DNA complex at 1.7 Å resolution and compare it with the known structure of the NgoMIV–DNA complex. We find that Ecl18kI flips both central nucleotides within the CCNGG sequence and buries the extruded bases in pockets within the protein. Nucleotide flipping disrupts Watson–Crick base pairing, induces a kink in the DNA and shifts the DNA register by 1 bp, making the distances between scissile phosphates in the Ecl18kI and NgoMIV cocrystal structures nearly identical. Therefore, the two enzymes can use a conserved DNA recognition module, yet recognize different sequences, and form superimposable dimers, yet generate different cleavage patterns. Hence, Ecl18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site. PMID:16628220

  16. Engineering lower inhibitor affinities in beta-D-xylosidase by site-directed mutagenesis of Trp 145

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta- D-xylosidase catalyzes hydrolysis of xylooligosaccharides to D-xylose monosaccharides. beta-Xylosidase from Selenomonas ruminantium, SXA, is the most active catalyst known for the reaction; however, its activity is inhibited by D-xylose and D-glucose (Ki values of ~10-2 M). Higher Ki’s could...

  17. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  18. Feature Matching with Affine-Function Transformation Models.

    PubMed

    Li, Hongsheng; Huang, Xiaolei; Huang, Junzhou; Zhang, Shaoting

    2014-12-01

    Feature matching is an important problem and has extensive uses in computer vision. However, existing feature matching methods support either a specific or a small set of transformation models. In this paper, we propose a unified feature matching framework which supports a large family of transformation models. We call the family of transformation models the affine-function family, in which all transformations can be expressed by affine functions with convex constraints. In this framework, the goal is to recover transformation parameters for every feature point in a template point set to calculate their optimal matching positions in an input image. Given pairwise feature dissimilarity values between all points in the template set and the input image, we create a convex dissimilarity function for each template point. Composition of such convex functions with any transformation model in the affine-function family is shown to have an equivalent convex optimization form that can be optimized efficiently. Four example transformation models in the affine-function family are introduced to show the flexibility of our proposed framework. Our framework achieves 0.0 percent matching errors for both CMU House and Hotel sequences following the experimental setup in [6]. PMID:26353148

  19. The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells

    PubMed Central

    Bhatia, Prateek A.; Moaddel, Ruin; Wainer, Irving W.

    2010-01-01

    CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodopetra frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp c-DNA, using a baculovirus expression system. The resulting CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [3H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9MRP1) column, etoposide and furosemide on the CMAC(Sf9MRP2) column and etoposide and fumitremorgin C on the CMAC(Sf9BCPR) column The binding affinities (Ki values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [3H]-etoposide on the CMAC(Sf9MRP1) column to a greater extent than (R)-verapamil and the relative IC50 values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC50 values were consistent with previously reported data. The results indicated that the CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system. PMID:20441926

  20. VLTI and KI Interferometric Observations of Massive Evolved Stars and Their Dusty Circumstellar Environments

    NASA Astrophysics Data System (ADS)

    Wallace, Debra J.; Danchi, W. C.; Rajagopal, J.; Chesneau, O.; Lopez, B.; Menut, J.; Monnier, J.; Tuthill, P.; Ireland, M.; Barry, R.; Richardson, L. J.

    2007-12-01

    Recent aperture-masking and interferometric observations of late-type WC Wolf-Rayet stars strongly support the theory that dust formation in these objects is a result of colliding winds in binary systems. To explore and quantify this possible explanation, we have conducted a high-resolution interferometric survey of late-type massive stars utilizing the VLTI, KI, IOTA, and FGS1r interferometers. We present here the motivation for this study. We also present the first results from the MIDI instrument on the VLTI, and the KI and IOTA observations. Our VLTI study is aimed primarily at resolving and characterizing the dust around the WC9 star WR 85a and the LBV WR 122, both dust-producing but at different phases of massive star evolution. Our IOTA and KI interferometric observations resolve the WR star WR 137 into a dust-producing binary system.

  1. Surface Structure of Kio (3) Grown By Heterogeneous Reaction of Ozone With Ki (001)

    SciTech Connect

    Brown, M.A.; Liu, Z.; Ashby, P.D.; Mehta, A.; Grimm, R.L.; Hemminger, J.C.

    2009-05-12

    The crystal structure of KIO{sub 3} grown by heterogeneous surface oxidation of KI (001) with ozone is reported. Under ambient reaction conditions (RH {approx}35%, room temperature) a thick layer of KIO{sub 3} grows at the gas-solid interface. Two doublets are present in the I(4d) X-ray photoelectron spectroscopy structure measurements, characteristic of unreacted KI (I{sup -}) from the substrate and the oxidized KIO{sub 3} (I{sup 5+}) reaction product. X-ray diffraction measurements confirm the presence at the interface of randomly oriented polycrystalline-triclinic KIO{sub 3} with an average particle diameter of 15 nm. KIO{sub 3} particle diameters determined from the X-ray diffraction peak widths are consistent with the results of atomic force microscopy. There is no X-ray powder diffraction evidence to suggest that the underlying KI substrate is altered in any manner during this heterogeneous interfacial reaction.

  2. Methods for Improving Aptamer Binding Affinity.

    PubMed

    Hasegawa, Hijiri; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers. PMID:27043498

  3. Identification of the Ki-1 antigen (CD30) as a novel therapeutic target in systemic mastocytosis

    PubMed Central

    Blatt, Katharina; Cerny-Reiterer, Sabine; Schwaab, Juliana; Sotlar, Karl; Eisenwort, Gregor; Stefanzl, Gabriele; Hoermann, Gregor; Mayerhofer, Matthias; Schneeweiss, Mathias; Knapp, Sylvia; Rülicke, Thomas; Hadzijusufovic, Emir; Bauer, Karin; Smiljkovic, Dubravka; Willmann, Michael; Reiter, Andreas; Horny, Hans-Peter

    2015-01-01

    The Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin lymphoma and anaplastic large-cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MCs) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MCs in advanced SM, and whether this surface structure may serve as therapeutic target in SM. As assessed by flow cytometry, CD30 was found to be expressed on the surface of neoplastic MCs in 3 of 25 patients (12%) with indolent SM, 4 of 7 patients (57%) with aggressive SM, and 4 of 7 patients (57%) with MC leukemia. The immature RAS-transformed human MC line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed MC line HMC-1.2 expressed no detectable CD30. The CD30-targeting antibody-conjugate brentuximab-vedotin inhibited proliferation in neoplastic MCs, with lower IC50 values obtained in CD30+ MCPV-1.1 cells (10 µg/mL) compared with CD30− HMC-1.2 cells (>50 µg/mL). In addition, brentuximab-vedotin suppressed the engraftment of MCPV-1.1 cells in NSG mice. Moreover, brentuximab-vedotin produced apoptosis in all CD30+ MC lines tested as well as in primary neoplastic MCs in patients with CD30+ SM, but did not induce apoptosis in neoplastic MCs in patients with CD30− SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE–induced histamine release in CD30+ MCs. Finally, brentuximab-vedotin and the KIT D816V-targeting drug PKC412 produced synergistic growth-inhibitory effects in MCPV-1.1 cells. Together, CD30 is a promising new drug target for patients with CD30+ advanced SM. PMID:26486787

  4. Effects of the KiVa antibullying program on cyberbullying and cybervictimization frequency among Finnish youth.

    PubMed

    Williford, Anne; Elledge, L Christian; Boulton, Aaron J; DePaolis, Kathryn J; Little, Todd D; Salmivalli, Christina

    2013-01-01

    Cyberbullying among school-aged children has received increased attention in recent literature. However, no empirical evidence currently exists on whether existing school-based antibullying programs are effective in targeting the unique aspects of cyberbullying. To address this important gap, the present study investigates the unique effects of the KiVa Antibullying Program on the frequency of cyberbullying and cybervictimization among elementary and middle school youth. Using data from a group randomized controlled trial, multilevel ordinal regression analyses were used to examine differences in the frequencies of cyberbullying and cybervictimization between intervention (N = 9,914) and control students (N = 8,498). The effects of age and gender on frequencies of cyber behaviors were also assessed across conditions. Results revealed a significant intervention effect on the frequency of cybervictimization; KiVa students reported lower frequencies of cybervictimization at posttest than students in a control condition. The effect of condition on the perpetration of cyberbullying was moderated by age. When student age was below the sample mean, KiVa students reported lower frequencies of cyberbullying than students in the control condition. We also found evidence of classroom level variation in cyberbullying and cybervictimization, suggesting cyberbullying is in part a classroom-level phenomenon. KiVa appears to be an efficacious program to address cyber forms of bullying and victimization. We discuss several unique aspects of KiVa that may account for the significant intervention effects. Results suggest that KiVa is an intervention option for schools concerned with reducing cyberbullying behavior and its deleterious effects on children's adjustment.

  5. Association analysis of polymorphisms in caprine KiSS1 gene with reproductive traits.

    PubMed

    Maitra, A; Sharma, Rekha; Ahlawat, Sonika; Tantia, M S; Roy, Manoranjan; Prakash, Ved

    2014-12-10

    KiSS1 is considered to be a key mediator of molecular mechanism of reproduction (puberty and prolificacy) in mammals. Kisspeptins are a family of structurally related peptides, encoded by KiSS1 gene, with ability to regulate gonadotropin-releasing hormone and hence hypothalamic-pituitary-gonadal axis. The present study investigated the polymorphism of caprine KiSS1 gene in 9 Indian goat breeds differing in sexual precocity and prolificacy. Comparison of KiSS1 amplified sequences of indigenous goats resulted in identification of nine SNPs (intron (1) G296C, T455G, T505A, T693C, T950C and intron (2) T1125C, A2510G, C2540T, A2803G) of which four are novel. These loci were not segregating together (r(2)<0.33). Mutations existed in both, sexually precocious and late-maturing goat breeds as well as low and high prolificacy goat breeds. Three loci reported to be associated with goat litter size (G296C, G2510A and C2540T) were identified in Indian goats as well. Association between loci of KiSS1 gene and age of puberty as well as litter size was explored in Black Bengal (N=158), a sexually precocious and prolific goat breed of India by designing PCR-RFLP. None of the mutations were found to be associated with reproductive traits however, difference in litter size as well age of sexual maturity for different genotypes indicates that the study on additional data based on more number of breeds and animals would be interesting to perform. Considering the importance of the reproductive trait in small ruminants, the results extend the limited information on genetic variation of the caprine KiSS1, which might contribute toward molecular breeding to enhance productivity of goat.

  6. Immunohistochemical Evaluation of p53 and Ki67 Expression in Skin Epithelial Tumors

    PubMed Central

    Khodaeiani, Effat; Fakhrjou, Ashraf; Amirnia, Mehdi; Babaei-nezhad, Shahla; Taghvamanesh, Farshid; Razzagh-Karimi, Elham; Alikhah, Hossein

    2013-01-01

    Background and Aims: The cellular mechanisms responsible for initiating or limiting the tumors including skin types are of great importance. The p53 is a tumor-inhibiting gene which is believed to be defective in many malignant situations. Ki67 is a non-histonic protein which is mainly interfere with the proliferation and has many controlling effects during the cell cycle. Because of their importance in skin tumor cell growth, this study aimed at evaluating the p53 and Ki67 expression in skin epithelial tumors by immunohistochemical method. Materials and Methods: In a descriptive setting, 50 biopsy samples (30 basal cell carcinomas (BCCs), 10 squamous cell carcinomas (SCCs), 8 keratoacanthomas (KAs), and 2 trichoepitheliomas (TEs)) were immunohistochemically evaluated for p53 and Ki67 expression during a 14-month period. The incidence and expression rate of these two variables were separately reported in each group of samples. Results: The expression rate of p53 was 67.77% for the BCCs, 50.20% for the SCCs, and null for the KAs. For both TEs, it was 50%. The expression rate of Ki67 was 57.33% for the BCCs, 47.70% for the SCCs, 37.5% for the KAs, and 0.0% for TEs. The incidence of P53+ cells was 100% and 90% in the BCC and SCC samples, respectively. The both TEs were positive in this regard. The incidence of Ki67+ cells was 100% for the BCC, SCC, and KA samples. The both TEs were negative in this regard. Conclusion: This study showed that the incidence rate of p53- and Ki67-positive cells is very high in skin malignant epithelial tumors. The expression rate of these two variables is comparable with reports in the literature. Further studies with large sample size are recommended to be carried out for KA and TE samples. PMID:23723466

  7. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  8. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    PubMed

    Brylinski, Michal

    2013-11-25

    A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely

  9. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    PubMed

    Brylinski, Michal

    2013-11-25

    A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely

  10. Searching for Galaxy Clusters in the VST-KiDS Survey

    NASA Astrophysics Data System (ADS)

    Radovich, M.; Puddu, E.; Bellagamba, F.; Moscardini, L.; Roncarelli, M.; Getman, F.; Grado, A.

    We present the methods and first results of the search for galaxy clusters in the Kilo Degree Survey (KiDS). The adopted algorithm and the criterium for selecting the member galaxies are illustrated. Here we report the preliminary results obtained over a small area (7 deg2), and the comparison of our cluster candidates with those found in the RedMapper and SZ Planck catalogues; the analysis to a larger area (148 deg2) is currently in progress. By the KiDS cluster search, we expect to increase the completeness of the clusters catalogue to z = 0.6-0.7 compared to RedMapper.

  11. Multiple GPCR conformations and signalling pathways: implications for antagonist affinity estimates

    PubMed Central

    Baker, Jillian G.; Hill, Stephen J.

    2007-01-01

    Antagonist affinity measurements have traditionally been considered important in characterizing the cell-surface receptors present in a particular cell or tissue. A central assumption has been that antagonist affinity is constant for a given receptor–antagonist interaction, regardless of the agonist used to stimulate that receptor or the downstream response that is measured. As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes. Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions. Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured). PMID:17629959

  12. Multiplexed protein profiling by sequential affinity capture

    PubMed Central

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter

    2016-01-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off‐target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi‐automated sequential capture assay. This novel bead‐based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read‐out by a secondary capture bead array. We demonstrate in a proof‐of‐concept setting that target detection via two sequential affinity interactions reduced off‐target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA‐based signal amplification, and demonstrate the applicability of the dual capture bead‐based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  13. Semiempirical Theories of the Affinities of Negative Atomic Ions

    NASA Technical Reports Server (NTRS)

    Edie, John W.

    1961-01-01

    The determination of the electron affinities of negative atomic ions by means of direct experimental investigation is limited. To supplement the meager experimental results, several semiempirical theories have been advanced. One commonly used technique involves extrapolating the electron affinities along the isoelectronic sequences, The most recent of these extrapolations Is studied by extending the method to Include one more member of the isoelectronic sequence, When the results show that this extension does not increase the accuracy of the calculations, several possible explanations for this situation are explored. A different approach to the problem is suggested by the regularities appearing in the electron affinities. Noting that the regular linear pattern that exists for the ionization potentials of the p electrons as a function of Z, repeats itself for different degrees of ionization q, the slopes and intercepts of these curves are extrapolated to the case of the negative Ion. The method is placed on a theoretical basis by calculating the Slater parameters as functions of q and n, the number of equivalent p-electrons. These functions are no more than quadratic in q and n. The electron affinities are calculated by extending the linear relations that exist for the neutral atoms and positive ions to the negative ions. The extrapolated. slopes are apparently correct, but the intercepts must be slightly altered to agree with experiment. For this purpose one or two experimental affinities (depending on the extrapolation method) are used in each of the two short periods. The two extrapolation methods used are: (A) an isoelectronic sequence extrapolation of the linear pattern as such; (B) the same extrapolation of a linearization of this pattern (configuration centers) combined with an extrapolation of the other terms of the ground configurations. The latter method Is preferable, since it requires only experimental point for each period. The results agree within

  14. [125I]AT-1012, a New High Affinity Radioligand for the α3β4 Nicotinic Acetylcholine Receptors

    PubMed Central

    Wu, Jinhua; Perry, David C.; Bupp, James E.; Jiang, Faming; Polgar, Willma E.; Toll, Lawrence; Zaveri, Nurulain T.

    2013-01-01

    Recent genetic and pharmacological studies have implicated the α3, β4 and a5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [125I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [125I]AT-1012 was characterized at the rat α3β4- and α4β2 nAChR transfected into HEK cells as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [125I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with a Bmax of 10.3 pmol/mg protein, similar to what was determined using [3H]epibatidine. Saturation isotherms suggested that [125I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [125I]AT-1012 at human α3β4α5 nAChR in a human α3β4aα nAChR transfected cell line. [125I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells, and [3H]epibatidine binding studies showed that AT-1012 had over 100-fold binding selectivity for the α3β4 over α4β2 nAChR. Ki values determined for known nAChR compounds using [125I]AT-1012 as radioligand were comparable to those obtained with [3H]epibatidine. [125I]AT-1012 was also used to label the α3β4 nAChR in rat brain slices in vitro using autoradiography which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We

  15. Affinity Proteomics in the mountains: Alpbach 2015.

    PubMed

    Taussig, Michael J

    2016-09-25

    The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. PMID:27118167

  16. Aptamers in Affinity Separations: Stationary Separation

    NASA Astrophysics Data System (ADS)

    Ravelet, Corinne; Peyrin, Eric

    The use of DNA or RNA aptamers as tools in analytical chemistry is a very promising field of research because of their capabilities to bind specifically the target molecules with an affinity similar to that of antibodies. Notably, they appear to be of great interest as target-specific ligands for the separation and capture of various analytes in affinity chromatography and related affinity-based methods such as magnetic bead technology. In this chapter, the recent developments of these aptamer-based separation/capture approaches are addressed.

  17. Phase I Hydroxylated Metabolites of the K2 Synthetic Cannabinoid JWH-018 Retain In Vitro and In Vivo Cannabinoid 1 Receptor Affinity and Activity

    PubMed Central

    Brents, Lisa K.; Reichard, Emily E.; Zimmerman, Sarah M.; Moran, Jeffery H.; Fantegrossi, William E.; Prather, Paul L.

    2011-01-01

    Background K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ9-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R). Methods/Principal Findings JWH-018, five potential monohydroxylated metabolites (M1–M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [3H]CP-55,940, and then for CB1R intrinsic efficacy using an [35S]GTPγS binding assay. JWH-018 and M1–M5 bound CB1Rs with high affinity, exhibiting Ki values that were lower than or equivalent to Δ9-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ9-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ9-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251. Conclusions/Significance Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher

  18. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes.

    PubMed

    Cuylen, Sara; Blaukopf, Claudia; Politi, Antonio Z; Müller-Reichert, Thomas; Neumann, Beate; Poser, Ina; Ellenberg, Jan; Hyman, Anthony A; Gerlich, Daniel W

    2016-06-29

    Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

  19. Draft Genome Sequence of the Moderately Halophilic Methanotroph Methylohalobius crimeensis Strain 10Ki

    PubMed Central

    Sharp, Christine E.; Smirnova, Angela V.; Kalyuzhnaya, Marina G.; Bringel, Françoise; Hirayama, Hisako; Jetten, Mike S. M.; Khmelenina, Valentina N.; Klotz, Martin G.; Knief, Claudia; Kyrpides, Nikos; Op den Camp, Huub J. M.; Reshetnikov, Alexander S.; Sakai, Yasuyoshi; Shapiro, Nicole; Trotsenko, Yuri A.; Vuilleumier, Stéphane; Woyke, Tanja

    2015-01-01

    Methylohalobius crimeensis strain 10Ki is a moderately halophilic aerobic methanotroph isolated from a hypersaline lake in the Crimean Peninsula, Ukraine. This organism has the highest salt tolerance of any cultured methanotroph. Here, we present a draft genome sequence of this bacterium. PMID:26067976

  20. Cyclin D1 and Ki-67 expression correlates to tumor staging in tongue squamous cell carcinoma

    PubMed Central

    de Carli, Marina-Lara; Sperandio, Felipe-Fornias; Hanemann, João-Adolfo-Costa; Pereira, Alessandro-Antônio-Costa

    2015-01-01

    Background The immunohistochemical expression of Cyclin D1 and Ki-67 were analyzed in tongue squamous cell carcinomas (SCC), relating them to the clinical and morphological exhibition of these tumors. Material and Methods Twenty-nine patients fulfilled the inclusion criteria; clinical data included gender, age, ethnicity and use of licit drugs such as alcohol and tobacco. The TNM staging and histopathological differentiation grading was assessed for each case. In addition, T1 patients were gathered with T2 patients; and T3 patients were gathered with T4 patients to assemble two distinct groups: (T1/T2) and (T3/T4). Results The mean follow-up time was 24 months and 30% of the patients died as a consequence of the disease, while 23.3% lived with the disease and 46.7% lived lesion-free. T1 and T2 tumors showed statistically lesser Ki-67 and Cyclin D1 staining when compared to T3 and T4 tumors. Conclusions Ki-67 and Cyclin D1 pose as auxiliary tools when determining the progression of tongue SCC at the time of diagnosis. Key words:Carcinoma, squamous cell, cyclin D, immunohistochemistry, Ki-67 antigen, prognosis. PMID:26449430

  1. Automated Ki-67 Quantification of Immunohistochemical Staining Image of Human Nasopharyngeal Carcinoma Xenografts

    PubMed Central

    Shi, Peng; Zhong, Jing; Hong, Jinsheng; Huang, Rongfang; Wang, Kaijun; Chen, Yunbin

    2016-01-01

    Nasopharyngeal carcinoma is one of the malignant neoplasm with high incidence in China and south-east Asia. Ki-67 protein is strictly associated with cell proliferation and malignant degree. Cells with higher Ki-67 expression are always sensitive to chemotherapy and radiotherapy, the assessment of which is beneficial to NPC treatment. It is still challenging to automatically analyze immunohistochemical Ki-67 staining nasopharyngeal carcinoma images due to the uneven color distributions in different cell types. In order to solve the problem, an automated image processing pipeline based on clustering of local correlation features is proposed in this paper. Unlike traditional morphology-based methods, our algorithm segments cells by classifying image pixels on the basis of local pixel correlations from particularly selected color spaces, then characterizes cells with a set of grading criteria for the reference of pathological analysis. Experimental results showed high accuracy and robustness in nucleus segmentation despite image data variance. Quantitative indicators obtained in this essay provide a reliable evidence for the analysis of Ki-67 staining nasopharyngeal carcinoma microscopic images, which would be helpful in relevant histopathological researches. PMID:27562647

  2. The National Heritage of Ki Hadjar Dewantara in Tamansiswa about Culture-Based Education and Learning

    ERIC Educational Resources Information Center

    Towaf, Siti Malikhah

    2016-01-01

    Global interdependence is a reality; in the security, economics, politics, socio-culture, and especially in the education of a nation;. Relevant to the need for an international dialog on education, this study tries to explore: 1) the concepts of culture-based education and learning of Ki Hadjar Dewantara (KHD) in Tamansiswa, 2) the results of…

  3. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes.

    PubMed

    Cuylen, Sara; Blaukopf, Claudia; Politi, Antonio Z; Müller-Reichert, Thomas; Neumann, Beate; Poser, Ina; Ellenberg, Jan; Hyman, Anthony A; Gerlich, Daniel W

    2016-07-14

    Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization. PMID:27362226

  4. The Role of Ki 'Im in Orchestrating Contrastive Focus in Biblical Hebrew

    ERIC Educational Resources Information Center

    Park, Grace Jeongyeon

    2011-01-01

    The so-called compound use of ki 'im has been interpreted mainly either with exceptive ("except" or "unless") or adversative meaning ("but" or "rather"), although it has sometimes also been interpreted in other ways such as "only" or "surely". These various meanings have been applied…

  5. Implementing the KiVa Antibullying Program: Recognition of Stable Victims

    ERIC Educational Resources Information Center

    Haataja, Anne; Sainio, Miia; Turtonen, Mira; Salmivalli, Christina

    2016-01-01

    Teachers do not always recognise students who are victimised by their peers. In this study, we examined the recognition of stable victims in 76 schools beginning to implement the KiVa antibullying programme. We focused on 348 victims (9-15 years) who reported victimisation at the pretest and still at wave 2, after five months of programme…

  6. KI-Aikido for Handicapped Students at Leeward Community College: Theory and Practice.

    ERIC Educational Resources Information Center

    MacGugan, Kirk

    In an effort to provide physical education instruction for handicapped students, Leeward Community College implemented, on a pilot basis, a non-credit course in KI-Aikido, an oriental martial art which combines theory and exercise toward the goal of controlling the body through the power of the mind. The course, offered to both handicapped and…

  7. Automated Ki-67 Quantification of Immunohistochemical Staining Image of Human Nasopharyngeal Carcinoma Xenografts.

    PubMed

    Shi, Peng; Zhong, Jing; Hong, Jinsheng; Huang, Rongfang; Wang, Kaijun; Chen, Yunbin

    2016-01-01

    Nasopharyngeal carcinoma is one of the malignant neoplasm with high incidence in China and south-east Asia. Ki-67 protein is strictly associated with cell proliferation and malignant degree. Cells with higher Ki-67 expression are always sensitive to chemotherapy and radiotherapy, the assessment of which is beneficial to NPC treatment. It is still challenging to automatically analyze immunohistochemical Ki-67 staining nasopharyngeal carcinoma images due to the uneven color distributions in different cell types. In order to solve the problem, an automated image processing pipeline based on clustering of local correlation features is proposed in this paper. Unlike traditional morphology-based methods, our algorithm segments cells by classifying image pixels on the basis of local pixel correlations from particularly selected color spaces, then characterizes cells with a set of grading criteria for the reference of pathological analysis. Experimental results showed high accuracy and robustness in nucleus segmentation despite image data variance. Quantitative indicators obtained in this essay provide a reliable evidence for the analysis of Ki-67 staining nasopharyngeal carcinoma microscopic images, which would be helpful in relevant histopathological researches. PMID:27562647

  8. Analysis of the Ki-67 index in the vaginal epithelium of castrated rats treated with tamoxifen

    PubMed Central

    Nery-Aguiar, Afif Rieth; Aguiar, Yousef Qathaf; Júnior, Airton Mendes Conde; Alencar, Airlane Pereira; Tavares, Cleciton Braga; Lopes-Costa, Pedro Vitor; Nazário, Afonso Celso; da Silva, Benedito Borges

    2016-01-01

    OBJECTIVES: Vaginal atrophy and breast cancer are common conditions in postmenopausal women and tamoxifen is the standard endocrine treatment for hormone-sensitive tumors. The present study aimed to assess the effect of tamoxifen on Ki-67 protein expression in the vaginal epithelium of castrated rats. MATERIAL AND METHODS: Forty Wistar-Hannover adult, virgin, castrated rats were randomly divided into two groups, group I (control, n=20) and group II (tamoxifen, n=20), receiving 0.5 ml of propylene glycol and 250 µg of tamoxifen diluted in 0.5 ml of propylene glycol, respectively, daily by gavage for 30 days. On the 31st day, the rats were euthanized and their vaginas were removed and fixed in 10% buffered formalin for the immunohistochemical study of Ki-67 protein expression. Data were analyzed by the Levene and Student's t tests (p<0.05). RESULTS: The mean index of Ki-67 expression in the rat vagina of groups I and II was 4.04±0.96 and 26.86±2.19, respectively (p<0.001). CONCLUSIONS: According to the results of the present study, tamoxifen, at the dose and treatment length used, induced a significant increase in the cell proliferation of the vaginal mucosa in castrated rats, as evaluated by Ki-67 protein expression. PMID:26934238

  9. PCNA, Ki-67 and p53 expressions in submandibular salivary gland tumours.

    PubMed

    Alves, F A; Pires, F R; De Almeida, O P; Lopes, M A; Kowalski, L P

    2004-09-01

    Salivary gland tumours are uncommon with a broad heterogeneity. The most common benign tumour is the pleomorphic adenoma, whereas mucoepidermoid carcinoma and adenoid cystic carcinoma predominate among the malignancies. Most salivary gland tumours occur in the parotid, and consequently clinical and biological data are normally derived from this site. This work describes the expressions of PCNA, Ki-67 and p53 in 15 pleomorphic adenomas, 15 mucoepidermoid carcinomas and 15 adenoid cystic carcinomas of the submandibular gland. Our results showed that all pleomorphic adenomas were negative for p53 and Ki-67 with 66.6% being positive for PCNA. Conversely, p53 was positive in 53% of the mucoepidermoid carcinomas and in 20% of the adenoid cystic carcinomas. Ki-67 was expressed in 47.7% of the mucoepidermoid carcinomas and 40% of the adenoid cystic carcinomas. All malignant tumours were positive for PCNA. These results indicate that the proliferative rate analysed with PCNA and Ki-67 and the expression of p53 in pleomorphic adenoma and adenoid cystic carcinoma of the submandibular gland were similar to those described in the parotid and minor salivary glands. However, mucoepidermoid carcinomas showed higher expression of these markers than those of other salivary glands. This work is the first describing the expression of these immunohistochemical markers exclusively in submandibular salivary gland tumours.

  10. Synthesis and Opioid Receptor Binding Affinities of 2-Substituted and 3-Aminomorphinans: Ligands for mu, kappa and delta Opioid Receptors

    PubMed Central

    Decker, Michael; Si, Yu-Gui; Knapp, Brian I.; Bidlack, Jean M.; Neumeyer, John L.

    2009-01-01

    The phenolic group of the potent μ and κ opioid morphinan agonist/antagonists cyclorphan and butorphan was replaced by phenylamino and benzylamino groups including compounds with p-substituents in the benzene ring. These compounds are highly potent μ and κ ligands, e. g. p-methoxyphenylaminocyclorphan showing a Ki of 0.026 nM at the mu and a Ki of 0.03 nM at the kappa receptor. Phenyl carbamates and phenylureas were synthesized and investigated. Selective o-formylation of butorphan and levorphanol was achieved. This reaction opened the way to a large set of 2-substituted 3-hydroxymorphinans, including 2-hydroxymethyl-, 2-aminomethyl-, and N-substituted 2-aminomethyl-3-hydroxymorphinans. Bivalent ligands bridged in the 2-position were also synthesized and connected with secondary and tertiary aminomethyl groups, amide bonds or hydroxymethylene groups, respectively. Although most of the 2-substituted morphinans showed considerably lower affinities compared to their parent compounds, the bivalent ligand approach led to significantly higher affinities compared to the univalent aminomethylmorphinans. PMID:19928862

  11. Isotope shift in the electron affinity of lithium

    SciTech Connect

    Bubin, Sergiy; Komasa, Jacek; Stanke, Monika; Adamowicz, Ludwik

    2009-12-21

    Very accurate electron affinity (EA) calculations of {sup 6}Li and {sup 7}Li (and {sup {infinity}L}i) have been performed using explicitly correlated Gaussian functions and a variational approach that explicitly includes the nuclear motion in the calculations (i.e., the approach that does not assume the Born-Oppenheimer approximation). The leading relativistic and quantum electrodynamics corrections to the electron affinities were also calculated. The results are the most accurate theoretical values obtained for the studied systems to date. Our best estimates of the {sup 7}Li and {sup 6}Li EAs are 4984.9842(30) and 4984.9015(30) cm{sup -1}, respectively, and of the {sup 7}Li/{sup 6}Li EA isotope shift is 0.0827 cm{sup -1}.

  12. Isotope shift in the electron affinity of lithium.

    PubMed

    Bubin, Sergiy; Komasa, Jacek; Stanke, Monika; Adamowicz, Ludwik

    2009-12-21

    Very accurate electron affinity (EA) calculations of (6)Li and (7)Li (and (infinity)Li) have been performed using explicitly correlated Gaussian functions and a variational approach that explicitly includes the nuclear motion in the calculations (i.e., the approach that does not assume the Born-Oppenheimer approximation). The leading relativistic and quantum electrodynamics corrections to the electron affinities were also calculated. The results are the most accurate theoretical values obtained for the studied systems to date. Our best estimates of the (7)Li and (6)Li EAs are 4984.9842(30) and 4984.9015(30) cm(-1), respectively, and of the (7)Li/(6)Li EA isotope shift is 0.0827 cm(-1).

  13. Molecular modeling of oscillating GHz electric field influence on the kinesin affinity to microtubule

    NASA Astrophysics Data System (ADS)

    R. Saeidi, H.; S. Setayandeh, S.; Lohrasebi, A.

    2015-08-01

    Kinesin is a microtubule-associated motor protein which can respond to the external electric field due to its polarity. Using a molecular dynamics simulation method, the effect of such a field on the affinity of kinesin to the αβ-tubulin is investigated in this study. To consider kinesin affinity, the system is exposed to an electric field of 0.03 V/nm with frequency values of 1, 2, …, 9, and 10 GHz. It is found that the applied electric field can change kinesin affinity to the microtubule. These changes could perturb the normal operation of kinesin, such as the processive motility of kinesin on the microtubule.

  14. The Plant-Derived Bauhinia bauhinioides Kallikrein Proteinase Inhibitor (rBbKI) Attenuates Elastase-Induced Emphysema in Mice.

    PubMed

    Martins-Olivera, Bruno Tadeu; Almeida-Reis, Rafael; Theodoro-Júnior, Osmar Aparecido; Oliva, Leandro Vilela; Neto Dos Santos Nunes, Natalia; Olivo, Clarice Rosa; Vilela de Brito, Marlon; Prado, Carla Máximo; Leick, Edna Aparecida; Martins, Mílton de Arruda; Oliva, Maria Luiza Vilela; Righetti, Renato Fraga; Tibério, Iolanda de Fátima Lopes Calvo

    2016-01-01

    Background. Elastase mediates important oxidative actions during the development of chronic obstructive pulmonary disease (COPD). However, few resources for the inhibition of elastase have been investigated. Our study evaluated the ability of the recombinant plant derived Bauhinia bauhinioides Kallikrein proteinase Inhibitor (rBbKI) to modulate elastase-induced pulmonary inflammation. Methods. C57Bl/6 mice were given intratracheal elastase (ELA group) or saline (SAL group) and were treated intraperitoneally with rBbKI (ELA-rBbKI and SAL-rBbKI groups). At day 28, the following analyses were performed: (I) lung mechanics, (II) exhaled nitric oxide (ENO), (III) bronchoalveolar lavage fluid (BALF), and (IV) lung immunohistochemical staining. Results. In addition to decreasing mechanical alterations and alveolar septum disruption, rBbKI reduced the number of cells in the BALF and decreased the cellular expression of TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS in airways and alveolar walls compared with the ELA group. rBbKI decreased the volume proportion of 8-iso-PGF2α, collagen, and elastic fibers in the airways and alveolar walls compared with the ELA group. A reduction in the number of MUC-5-positive cells in the airway walls was also observed. Conclusion. rBbKI reduced elastase-induced pulmonary inflammation and extracellular matrix remodeling. rBbKI may be a potential pharmacological tool for COPD treatment. PMID:27528793

  15. The Plant-Derived Bauhinia bauhinioides Kallikrein Proteinase Inhibitor (rBbKI) Attenuates Elastase-Induced Emphysema in Mice.

    PubMed

    Martins-Olivera, Bruno Tadeu; Almeida-Reis, Rafael; Theodoro-Júnior, Osmar Aparecido; Oliva, Leandro Vilela; Neto Dos Santos Nunes, Natalia; Olivo, Clarice Rosa; Vilela de Brito, Marlon; Prado, Carla Máximo; Leick, Edna Aparecida; Martins, Mílton de Arruda; Oliva, Maria Luiza Vilela; Righetti, Renato Fraga; Tibério, Iolanda de Fátima Lopes Calvo

    2016-01-01

    Background. Elastase mediates important oxidative actions during the development of chronic obstructive pulmonary disease (COPD). However, few resources for the inhibition of elastase have been investigated. Our study evaluated the ability of the recombinant plant derived Bauhinia bauhinioides Kallikrein proteinase Inhibitor (rBbKI) to modulate elastase-induced pulmonary inflammation. Methods. C57Bl/6 mice were given intratracheal elastase (ELA group) or saline (SAL group) and were treated intraperitoneally with rBbKI (ELA-rBbKI and SAL-rBbKI groups). At day 28, the following analyses were performed: (I) lung mechanics, (II) exhaled nitric oxide (ENO), (III) bronchoalveolar lavage fluid (BALF), and (IV) lung immunohistochemical staining. Results. In addition to decreasing mechanical alterations and alveolar septum disruption, rBbKI reduced the number of cells in the BALF and decreased the cellular expression of TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS in airways and alveolar walls compared with the ELA group. rBbKI decreased the volume proportion of 8-iso-PGF2α, collagen, and elastic fibers in the airways and alveolar walls compared with the ELA group. A reduction in the number of MUC-5-positive cells in the airway walls was also observed. Conclusion. rBbKI reduced elastase-induced pulmonary inflammation and extracellular matrix remodeling. rBbKI may be a potential pharmacological tool for COPD treatment.

  16. IDH1/2 mutation status combined with Ki-67 labeling index defines distinct prognostic groups in glioma.

    PubMed

    Zeng, Ailiang; Hu, Qi; Liu, Yanwei; Wang, Zheng; Cui, Xiaoming; Li, Rui; Yan, Wei; You, Yongping

    2015-10-01

    The current World Health Organization (WHO) classification of human gliomas is mainly based on morphology. However, it has limitations in prognostic prediction. We examined whether combining isocitrate dehydrogenase (IDH) 1/2 mutation status with the Ki-67 labeling index would improve the definition of prognostically distinct entities. We investigated the correlation of Ki-67 expression with IDH1/2 mutation status and their impact on clinical outcome in 703 gliomas. Low Ki-67 expression closely overlapped with IDH1/2 mutation in our cohort (P < 0.0001). Patients with IDH1/2 mutation survived significantly longer than patients with wild-type IDH1/2 did (P < 0.0001); higher Ki-67 expression was associated with shorter progression-free survival and overall survival (OS) (P < 0.0001). IDH1/2 combined with Ki-67 was used to re-classify glioma patients into five groups. IDH1/2 mutant patients with low and moderate Ki-67 expression (Group1) had the best prognosis, whereas patients with wild-type IDH1/2 and high Ki-67 expression (Group5) had the worst prognosis (Median OS = 1527 vs. 355 days, P < 0.0001). To summarize, our new classification model distinguishes biologically distinct subgroups and provides prognostic information regardless of the conventional WHO grade. Classification based on IDH1/2 mutation status and Ki-67 expression level could be more convenient for clinical application and guide personalized treatment in malignant gliomas.

  17. The Plant-Derived Bauhinia bauhinioides Kallikrein Proteinase Inhibitor (rBbKI) Attenuates Elastase-Induced Emphysema in Mice

    PubMed Central

    Martins-Olivera, Bruno Tadeu; Theodoro-Júnior, Osmar Aparecido; Oliva, Leandro Vilela; Neto dos Santos Nunes, Natalia; Olivo, Clarice Rosa; Vilela de Brito, Marlon; Prado, Carla Máximo; Leick, Edna Aparecida; Martins, Mílton de Arruda

    2016-01-01

    Background. Elastase mediates important oxidative actions during the development of chronic obstructive pulmonary disease (COPD). However, few resources for the inhibition of elastase have been investigated. Our study evaluated the ability of the recombinant plant derived Bauhinia bauhinioides Kallikrein proteinase Inhibitor (rBbKI) to modulate elastase-induced pulmonary inflammation. Methods. C57Bl/6 mice were given intratracheal elastase (ELA group) or saline (SAL group) and were treated intraperitoneally with rBbKI (ELA-rBbKI and SAL-rBbKI groups). At day 28, the following analyses were performed: (I) lung mechanics, (II) exhaled nitric oxide (ENO), (III) bronchoalveolar lavage fluid (BALF), and (IV) lung immunohistochemical staining. Results. In addition to decreasing mechanical alterations and alveolar septum disruption, rBbKI reduced the number of cells in the BALF and decreased the cellular expression of TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS in airways and alveolar walls compared with the ELA group. rBbKI decreased the volume proportion of 8-iso-PGF2α, collagen, and elastic fibers in the airways and alveolar walls compared with the ELA group. A reduction in the number of MUC-5-positive cells in the airway walls was also observed. Conclusion. rBbKI reduced elastase-induced pulmonary inflammation and extracellular matrix remodeling. rBbKI may be a potential pharmacological tool for COPD treatment. PMID:27528793

  18. Expression of bcl-2, p53 and Ki-67 in arsenical skin cancers.

    PubMed

    Chang, C H; Tsai, R K; Chen, G S; Yu, H S; Chai, C Y

    1998-10-01

    To investigate the regulation of apoptosis and proliferation in arsenic-induced skin cancers, we examined the expression of bcl-2, p53, and Ki-67 using immunohistochemical staining. Thirty patients with Bowen's disease (BD), ten with basal cell carcinoma (BCC), eight with squamous cell carcinoma (SCC) and eleven of perilesional normal skin (PLN) of the non-sun exposure sites from endemic area were examined. The results showed that: 1) bcl-2 was expressed in all of the BCC homogeneously, in none of the SCC, and in 12/30 of the BD focally or homogeneously; 2) p53 was expressed in all of the arsenical skin cancers with a labelling index of 75 +/- 14% of BD, 50 +/- 17% of BCC, 61 +/- 15% of SCC, and also in all of the perilesional normal skin with a labelling index of 55 +/- 24%; 3) Ki-67 was expressed in all of the skin cancers with labelling index of 58 +/- 17% of BD, 12 +/- 7% of BCC, 47 +/- 21% of SCC, and in 9/11 of PLN with a labelling index of 41 +/- 24%. Expression of bcl-2 in BCC or BD is related to the phenotype of germinative basal cell. The constant expression of bcl-2 i early dysplastic cells of BD and the earliest expression of P53 in the basal cells of perilesional normal skin indicate that the initial step of arsenic-induced carcinogenesis is from the basal germinative cells. There is no mutual relationship between bcl-2, p53 or Ki-67 expression in any type of the arsenical skin cancers, but there is a positive correlation between p53 and Ki-67 expression identified in perilesional normal skin. BD had the highest labelling index of p53 and Ki-67.

  19. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  20. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  1. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  2. Designing Chaotic Systems by Piecewise Affine Systems

    NASA Astrophysics Data System (ADS)

    Wu, Tiantian; Li, Qingdu; Yang, Xiao-Song

    Based on mathematical analysis, this paper provides a methodology to ensure the existence of homoclinic orbits in a class of three-dimensional piecewise affine systems. In addition, two chaotic generators are provided to illustrate the effectiveness of the method.

  3. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  4. [Prevalence of common allergies in children and adolescents in Germany: results of the KiGGS study: first follow-up (KiGGS Wave 1)].

    PubMed

    Schmitz, R; Thamm, M; Ellert, U; Kalcklösch, M; Schlaud, M

    2014-07-01

    The first follow-up of the German Health Interview and Examination Survey for Children and Adolescents (KiGGS Wave 1) was conducted from 2009 to 2012 as a combined cross-sectional and longitudinal study and provides, among other things, data on allergic diseases. Data collection was carried out by telephone interviews. In total, 6,093 girls and 6,275 boys were included, among them 4,455 newly recruited 0- to 6-year-olds (response 38.8%) and 7,913 KiGGS follow-up participants aged 7-17 years (response 72.9%). Based on parent reports, 15.6% (95% confidence interval 14.7-16.5) of children and adolescents aged 0-17 years were currently affected by at least one atopic disease. The 12-month prevalence rates of hay fever, atopic dermatitis, and asthma were 9.1% (8.4-9.8), 6.0% (5.4-6.6), and 4.1% (3.6-4.6), respectively. In all, 2.2% (1.9-2.6) of the children and adolescents were currently suffering from contact dermatitis. Compared with the baseline KiGGS survey from 2003 to 2006, a higher percentage of participants reported the occurrence of asthma within the past 12 months in the recent KiGGS Wave 1 (4.1 vs. 3.2%; p = 0.0034). The total increase is mainly due to higher prevalence rates among 0- to 6-year-olds, especially in girls. Higher 12-month prevalence rates can be also observed for hay fever among 0- to 6-year-olds, especially in girls, although the total increase is not statistically significant (9.1 vs. 8.3%; p = 0.08). There was a declining trend for atopic dermatitis: 6.8% (2003-2006) vs. 5.4% (2009-2012); p = 0.0015. PMID:24950826

  5. [Chronic and vaccine-preventable diseases in children and adolescents in Germany: results of the KiGGS study: first follow up (KiGGS wave 1)].

    PubMed

    Neuhauser, H; Poethko-Müller, C

    2014-07-01

    The German Health Interview and Examination Survey for Children and Adolescents (KiGGS) 2003-2006 is the first nationwide comprehensive study on the health of children and adolescents living in Germany. The KiGGS first interview follow-up is a telephone interview study that collected, among other things, data on a number of chronic and vaccine-preventable diseases in 2009-2012 and is a combined cross-sectional and longitudinal study based on a population registry sample from the 167 KiGGS study points. The analysis is based on 12,368 respondents (7913 KiGGS follow-up participants aged 7-17 years, response 72 % and 4455 newly recruited 0- to 6-year-olds, response 42 %). Based on parent reports the lifetime prevalence of both chickenpox and pertussis has decreased in the population targeted by recently changed vaccination recommendations. For measles the prevalence remained unsatisfactorily high in each investigated age group. Of the children and adolescents aged 0-17 years 16 % (95 % confidence interval CI 15.2-17.0 %) had a long-standing chronic health condition according to the parents. Of these, however, only one in five was affected in their routine daily activities. The lifetime prevalence in 7- to 17-year-olds was 1.2 % (0.9-1.6) for epilepsy (0.4 % for the past 12 months), 5.0 % (4.4-5.7) for migraine, 0.2 % (0.1-0.3) for diabetes and in 0 to 6-year-olds 2.0 % (1.5-2.6) for heart conditions and 3.1 % (2.5-3.8) for febrile seizures with a -prevalence in 0 to 2-year-olds which are most affected of 1.0 % (0.6-1.6) in the past 12 months. The vast majority of children and adolescents in Germany are in good or very good health as suggested by other results reported in this issue; however, chronic conditions are not rare and need continuous monitoring. These results confirm that implementation of the vaccination recommendations of the German Standing Committee on Vaccination (STIKO) can lead to effective prevention of infectious diseases in Germany.

  6. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

    PubMed

    Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S

    2016-01-01

    Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924

  7. [76Br]BMK-152, a non-peptide analogue, with high affinity and low non-specific binding for the Corticotropin-Releasing Factor Type 1 Receptor (CRF1 receptor)

    PubMed Central

    Jagoda, Elaine M.; Lang, Lixin; McCullough, Karen; Contoreggi, Carlo; Kim, B. Moon; Ma, Ying; Rice, Kenner C.; Szajek, Lawrence P; Eckelman, William C.; Kiesewetter, Dale O.

    2013-01-01

    Corticotropin-releasing factor (CRF), a neuropeptide, regulates endocrine and autonomic responses to stress through G-protein coupled receptors, CRF1 or CRF2. A PET ligand able to monitor changes in CRF1 receptor occupancy in vivo would aid in understanding the pathophysiology of stress related diseases as well as in the clinical development of non-peptide antagonists with therapeutic value. We have radiolabeled the CRF1 receptor ligand, BMK-152 ([8-(4-bromo-2,6-dimethoxyphenyl)-2,7-dimethylpyrazolo[1,5-α][1,3,5]triazin-4-yl]-N,N-bis-(2-methoxyethyl)amine; ClogP= 2.6), at both the 3 and 4 position with [76Br]. Using in vitro autoradiography saturation studies the 4-[76Br]BMK-152 exhibited high affinity binding to both rat (Kd = 0.23 ± 0.07 nM; n=3) and monkey frontal cortex (Kd = 0.31 ± 0.08 nM; n=3) consistent with CRF1 receptor regional distribution whereas with the 3-[76Br]BMK-152, the Kd's could not be determined due to high non-specific binding. In vitro autoradiography competition studies using [125I]Tyr0-o-CRF confirmed that 3-Br-BMK-152 (Ki = 24.4 ± 4.9 nM; n=3) had lower affinity (70 fold) than 4-Br-BMK-152 (Ki = 0.35 ± 0.07 nM; n=3) in monkey frontal cortex and similiar studies using [125I]Sauvagine confirmed CRF1 receptor selectivity. In vivo studies with P-glycoprotein (PGP) knockout mice (KO) and their wildtype littermates (WT) showed that the brain uptake of 3-[76Br]BMK/4-[76Br]BMK was increased < 2 fold in KO vs WT indicating that 3-[76Br]BMK-152/4-[76Br]BMK was not a Pgp substrate. Rat brain uptakes of 4-[76Br] BMK-152 from ex vivo autoradiography studies showed regional localization consistent with known published CRF1 receptor distribution and potential as a PET ligand for in vivo imaging of CRF1 receptors. PMID:21308801

  8. Predicting Lymph Node Metastasis in Endometrial Cancer Using Serum CA125 Combined with Immunohistochemical Markers PR and Ki67, and a Comparison with Other Prediction Models

    PubMed Central

    Xue, Xiaohong; Wang, Huaying; Shan, Weiwei; Ning, Chengcheng; Zhou, Qiongjie; Chen, Xiaojun; Luo, Xuezhen

    2016-01-01

    We aimed to evaluate the value of immunohistochemical markers and serum CA125 in predicting the risk of lymph node metastasis (LNM) in women with endometrial cancer and to identify a low-risk group of LNM. The medical records of 370 patients with endometrial endometrioid adenocarcinoma who underwent surgical staging in the Obstetrics & Gynecology Hospital of Fudan University were collected and retrospectively reviewed. Immunohistochemical markers were screened. A model using serum cancer antigen 125 (CA125) level, the immunohistochemical markers progesterone receptor (PR) and Ki67 was created for prediction of LNM. A predicted probability of 4% among these patients was defined as low risk. The developed model was externally validated in 200 patients from Shanghai Cancer Center. The efficiency of the model was compared with three other reported prediction models. Patients with serum CA125 < 30.0 IU/mL, either or both of positive PR staining > 50% and Ki67 < 40% in cancer lesion were defined as low risk for LNM. The model showed good discrimination with an area under the receiver operating characteristic curve of 0.82. The model classified 61.9% (229/370) of patients as being at low risk for LNM. Among these 229 patients, 6 patients (2.6%) had LNM and the negative predictive value was 97.4% (223/229). The sensitivity and specificity of the model were 84.6% and 67.4% respectively. In the validation cohort, the model classified 59.5% (119/200) of patients as low-risk, 3 out of these 119 patients (2.5%) has LNM. Our model showed a predictive power similar to those of two previously reported prediction models. The prediction model using serum CA125 and the immunohistochemical markers PR and Ki67 is useful to predict patients with a low risk of LNM and has the potential to provide valuable guidance to clinicians in the treatment of patients with endometrioid endometrial cancer. PMID:27163153

  9. MCDHF calculation of electron affinities of Group I and Group IB atomic anions

    NASA Astrophysics Data System (ADS)

    Li, Junqin; Zhao, Zilong; Zhang, Xuemei

    2014-08-01

    The affinities of negative ions for elements of Group I and Group IB have been calculated using the multi-configuration Dirac-Hartree-Fock (MCDHF) method. The difference between the total energy of the ground state of the atom and that of its anion is used to obtain the electron affinity. The theoretical results for these elements agree well with measured values, and have a deviation less than 0.5% with respect to measured values for most of the elements. With a systematic calculation method, this work gives a high-accuracy theoretical value for the electron affinities of the elements of Group I and Group IB. For element Fr, there is no experimental value.

  10. Affinity learning with diffusion on tensor product graph.

    PubMed

    Yang, Xingwei; Prasad, Lakshman; Latecki, Longin Jan

    2013-01-01

    In many applications, we are given a finite set of data points sampled from a data manifold and represented as a graph with edge weights determined by pairwise similarities of the samples. Often the pairwise similarities (which are also called affinities) are unreliable due to noise or due to intrinsic difficulties in estimating similarity values of the samples. As observed in several recent approaches, more reliable similarities can be obtained if the original similarities are diffused in the context of other data points, where the context of each point is a set of points most similar to it. Compared to the existing methods, our approach differs in two main aspects. First, instead of diffusing the similarity information on the original graph, we propose to utilize the tensor product graph (TPG) obtained by the tensor product of the original graph with itself. Since TPG takes into account higher order information, it is not a surprise that we obtain more reliable similarities. However, it comes at the price of higher order computational complexity and storage requirement. The key contribution of the proposed approach is that the information propagation on TPG can be computed with the same computational complexity and the same amount of storage as the propagation on the original graph. We prove that a graph diffusion process on TPG is equivalent to a novel iterative algorithm on the original graph, which is guaranteed to converge. After its convergence we obtain new edge weights that can be interpreted as new, learned affinities. We stress that the affinities are learned in an unsupervised setting. We illustrate the benefits of the proposed approach for data manifolds composed of shapes, images, and image patches on two very different tasks of image retrieval and image segmentation. With learned affinities, we achieve the bull's eye retrieval score of 99.99 percent on the MPEG-7 shape dataset, which is much higher than the state-of-the-art algorithms. When the data

  11. Proton Affinity of Isomeric Dipeptides Containing Lysine and Non-Proteinogenic Lysine Homologues.

    PubMed

    Batoon, Patrick; Ren, Jianhua

    2016-08-18

    Conformational effects on the proton affinity of oligopeptides have been studied using six alanine (A)-based acetylated dipeptides containing a basic probe that is placed closest to either the C- or the N-terminus. The basic probe includes Lysine (Lys) and two nonproteinogenic Lys-homologues, ornithine (Orn) and 2,3-diaminopropionic acid (Dap). The proton affinities of the peptides have been determined using the extended Cooks kinetic method in a triple quadrupole mass spectrometer. Computational studies have been carried out to search for the lowest energy conformers and to calculate theoretical proton affinities as well as various molecular properties using the density functional theory. The dipeptides containing a C-terminal probe, ALys, AOrn, and ADap, were determined to have a higher proton affinity by 1-4 kcal/mol than the corresponding dipeptides containing an N-terminal probe, LysA, OrnA, and DapA. For either the C-probe peptides or the N-probe peptides, the proton affinity reduces systematically as the side-chain of the probe residue is shortened. The difference in the proton affinities between isomeric peptides is largely associated with the variation of the conformations. The peptides with higher values of the proton affinity adopt a relatively compact conformation such that the protonated peptides can be stabilized through more efficient internal solvation. PMID:27459294

  12. Prognostic relevance of a novel proliferation marker, Ki-S11, for soft-tissue sarcoma. A multivariate study.

    PubMed Central

    Rudolph, P.; Kellner, U.; Chassevent, A.; Collin, F.; Bonichon, F.; Parwaresch, R.; Coindre, J. M.

    1997-01-01

    In 132 soft-tissue sarcomas and 52 benign soft-tissue tumors, cellular proliferation was examined by immunohistochemistry using monoclonal antibodies Ki-S11 (Ki-67 antigen) and Ki-S1 (topoisomerase II alpha) and by flow cytometric analysis of the S-phase fraction (SPF). Malignant tumors were graded histologically according to the Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC) system. Patient age, sex, tumor location, histological type, and DNA ploidy were considered as additional prognostic variables. Consistent immunoreactivity was seen in approximately 95% of the cases, and determination of SPF was possible in approximately 60% Ki-S11 and Ki-S1 immunolabeling indices correlated in a linear manner. All proliferation parameters yielded significant differences between benign and malignant tumors. Ki-S11 and Ki-S1 immunoreactive scores also co-varied significantly with SPF, mitotic count, and histopathological grade. In univariate analysis, immunohistochemical proliferation indices, histopathological grade, mitotic count, and SPF were predictive of overall survival and the development of metastases. In multivariate analysis, immunolabeling scores of proliferation markers, grade, and SPF emerged as independent predictors of global survival and systemic progression. We conclude that the immunohistochemical assessment of proliferation, being more readily performable and more easily assessable than the equally relevant S phase fraction, may add appreciable information to the current prognostic models for soft-tissue sarcoma. Images Figure 1 Figure 2 Figure 3 PMID:9176393

  13. Targeted light-inactivation of the Ki-67 protein using theranostic liposomes leads to death of proliferating cells

    NASA Astrophysics Data System (ADS)

    Rahmanzadeh, Ramtin; Rai, Prakash; Gerdes, Johannes; Hasan, Tayyaba

    2010-02-01

    Nanomedicine is beginning to impact the treatment of several diseases and current research efforts include development of integrated nano-constructs (theranostics) which serve as probes for imaging and therapy in addition to delivering macromolecules intracellularly. In cancer, there is a vital unmet need for effective alternative treatments with high specificity and low systemic toxicity. This can be achieved by targeting key molecular markers associated with cancer cells with reduced effective drug doses. Here, we show an innovative proof-of-principle approach for efficient killing of proliferating ovarian cancer cells by inactivating a protein associated with cell proliferation namely, the nuclear Ki-67 protein (pKi-67), using nanotechnology-based photodynamic therapy (PDT). Antibodies against pKi-67 are widely used as prognostic tools for tumor diagnosis. In this work, anti pKi-67 antibodies were first conjugated to fluorescein isothiocyanate (FITC) and then encapsulated inside liposomes. After incubation of OVCAR-5 ovarian cancer cells with these liposomes, confocal microscopy confirmed the localization of the antibodies to the nucleoli of the cells. Irradiation with a 488 nm laser led to a significant loss of cell viability. The specificity of this approach for pKi-67 positive cells was demonstrated in confluent human lung fibroblasts (MRC-5) where only a small population of cells stain positive for pKi-67 and only minimal cell death was observed. Taken together, our findings suggest that pKi-67 targeted with nano-platform is an attractive therapeutic target in cancer therapy.

  14. Ki-67 index guided selection of preoperative chemotherapy for HER2-positive breast cancer: a randomized phase II trial.

    PubMed

    Yamaguchi, Takeshi; Mukai, Hirofumi

    2012-12-01

    Changes in Ki-67 may be a useful predictor of efficacy for preoperative therapy in breast cancer. This randomized Phase II trial will compare standard preoperative chemotherapy comprising paclitaxel and trastuzumab with Ki-67 index guided preoperative chemotherapy in patients with human epidermal growth factor receptor 2-positive operable breast cancer. In the Ki-67 index guided therapy, paclitaxel and trastuzumab were administered initially and the Ki-67 index is evaluated from biopsied specimens after 2 weeks of preoperative chemotherapy. The subsequent chemotherapy regimen is modified according to changes in the Ki-67 index from the start of therapy. If the Ki-67 index is reduced as expected, paclitaxel and trastuzumab are continued. If the Ki-67 index is not reduced as expected, the chemotherapy regimen is changed to epirubicin, cyclophosphamide and trastuzumab. The primary endpoint is the rate of pathological complete response. The secondary endpoints are the objective response rate, disease-free survival and overall survival. Two hundred patients were planned to be accrued.

  15. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  16. BC(50): a generalized, unifying affinity descriptor.

    PubMed

    Vacca, Alberto; Francesconi, Oscar; Roelens, Stefano

    2012-12-01

    Assessing binding affinities is an unavoidable step that we come across any time interactions between binding species are investigated. A quantitative evaluation of binding affinities relies on the determination of binding constants but, whilst the binding constant fully defines the affinity of a reagent for a ligand when only one complex species is formed, the same is not true when the interacting partners form more than one complex of different stoichiometry, because all complexes contribute to the overall binding affinity. Unfortunately, this situation is the rule rather than the exception in chemical systems, but a generally accepted solution for this issue has not yet been settled. In this Personal Account, we describe the evolution, from the initial idea to a fully developed stage, of a binding descriptor that has been developed with the aim of filling this gap, thereby providing scientists in all fields of chemistry with a unifying tool for the assessment of binding affinities based on the knowledge of the binding constants in systems that involve any number of complex species.

  17. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  18. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  19. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  20. KiSS-1: a likely candidate for the photoperiodic control of reproduction in seasonal breeders.

    PubMed

    Revel, Florent G; Saboureau, Michel; Masson-Pévet, Mireille; Pévet, Paul; Mikkelsen, Jens D; Simonneaux, Valérie

    2006-01-01

    In seasonal species, photoperiod exerts tight regulation of reproduction to ensure that birth occurs at the most favorable time of yr. A distinct photoneuroendocrine circuit composed of the retina, suprachiasmatic nucleus (SCN) of the hypothalamus, and pineal gland transduces daylength into a rhythmic secretion of melatonin. The duration of the night-time rise of this hormone conveys daylength information to the organism. Melatonin is known to mediate the control of seasonal reproduction, but how it modulates sexual activity is far from understood. Recent data indicate that the product of the KiSS-1 gene is a potent stimulator of the hypothalamic-pituitary-gonadal axis and may play, together with its receptor GPR54, a central role in the neuroendocrine regulation of gonadotropin secretion. This article briefly reviews these findings and presents arguments that KiSS-1 could take part in the seasonal control of reproduction.

  1. Neoplastic transformation of a human prostate epithelial cell line by the v-Ki-ras oncogene.

    PubMed

    Parda, D S; Thraves, P J; Kuettel, M R; Lee, M S; Arnstein, P; Kaighn, M E; Rhim, J S; Dritschilo, A

    1993-01-01

    Investigations of mechanisms of human prostate carcinogenesis are limited by the unavailability of a suitable in vitro model system. We have demonstrated that an immortal, but nontumorigenic, human epithelial cell line (267B1) established from fetal prostate tissue can be malignantly transformed by a biological carcinogen, and can serve as a useful model for investigations of the progression steps of carcinogenesis. Activated Ki-ras was introduced into 267B1 cells by infection with the Kirsten murine sarcoma virus. Morphological alterations and anchorage-independent growth were observed; when cells were injected into nude mice, poorly differentiated adenocarcinomas developed. These findings represent the first evidence of malignant transformation of human prostate epithelial cells in culture, and support a role for Ki-ras activation in a multistep process for prostate neoplastic transformation.

  2. Valuing Essays: Essaying Values

    ERIC Educational Resources Information Center

    Badley, Graham

    2010-01-01

    The essay regularly comes under attack. It is criticised for being rigidly linear rather than flexible and reflective. I first challenge this view by examining reasons why the essay should be valued as an important genre. Secondly, I propose that in using the essay form students and academics necessarily exemplify their own critical values. Essays…

  3. The Nishino Breathing Method and Ki-energy (Life-energy): A Challenge to Traditional Scientific Thinking

    PubMed Central

    Ohnishi, S. Tsuyoshi; Ohnishi, Tomoko

    2006-01-01

    The breathing method, which was developed and is being taught by Kozo Nishino, a Japanese Ki-expert, is for raising the levels of Ki-energy (life-energy or the vitality) of an individual. It is neither a therapy nor a healing technique. However, many of his students have experienced an improvement in their health, and in some cases, they were able to overcome health problems by themselves. Since this is an interesting subject from the standpoint of complementary and alternative medicine (CAM), we have been collaborating with Nishino to conduct a scientific investigation of his Ki-energy. We found that Nishino's Ki-energy can inhibit cell division of cancer cells, protect isolated mitochondria from heat deterioration and reduce lipid peroxidation in heat-treated mitochondria. Although Ki-energy may consist of several different energy forms, we found that at least one of them is near-infrared radiation between the wavelength range of 0.8 and 2.7 µm. Another interesting observation at his school is the Taiki-practice (paired Ki-practice). During this practice, Nishino can ‘move’ his students without any physical contact. Many of them run, jump or roll on the floor when they receive his Ki-energy. We studied this and propose that ‘information’ is conveyed through the air between two individuals by Ki-energy. This may be called a five sense-independent, life-to-life communication by Ki. All of our results suggest that we should re-evaluate the Cartesian dualism (separation of mind and body) which has been a fundamental principle of modern science for the past three centuries. PMID:16786048

  4. Relationship between tumour shrinkage and reduction in Ki67 expression after primary chemotherapy in human breast cancer

    PubMed Central

    Bottini, A; Berruti, A; Bersiga, A; Brizzi, M P; Bruzzi, P; Aguggini, S; Brunelli, A; Bolsi, G; Allevi, G; Generali, D; Betri, E; Bertoli, G; Alquati, P; Dogliotti, L

    2001-01-01

    The association between tumour shrinkage and reduction in kinetic cell activity after primary chemotherapy in human breast cancer is still a matter of investigation. 157 patients with T2-4, N0-1, M0 breast cancer received primary chemotherapy consisting of either the CMF regimen + tamoxifen (the first consecutive 76 cases) or the single agent epirubicin (the subsequent 81). Ki67, p53, bcl2, c-erbB2 and steroid hormone receptors were evaluated immunohistochemically in tumour specimens obtained before chemotherapy and at surgery. Tumour shrinkage of >50% occurred in 72.4% of patients. Ki67 expression significantly decreased after chemotherapy; the reduction correlated with tumour response in both univariate (P < 0.005) and multivariate analysis (P = 0.02). p53, bcl-2, steroid hormone receptor and c-erbB2 immunostaining were scarcely affected. Baseline bcl2 (P = 0.04) and c-erbB2 (P = 0.02) were directly and inversely associated with the reduction in Ki67 immunostaining, respectively. Baseline p53 expression (P < 0.01) was directly related with Ki67 expression at residual tumour, whereas oestrogen receptor expression (P < 0.001) was inversely related. Ki67 at residual tumour was a better predictor for relapse-free survival (RFS) than baseline Ki67. Clinical response (P < 0.03), but not reduction in Ki67, was a significant independent predictor for disease recurrence. Chemotherapy was found to induce tumour shrinkage and to reduce the number of cells in the cell cycle, but its effect on tumour biology/aggressiveness was minimal. Reduction in Ki67 immunostaining correlated with clinical response but failed to be related to RFS. Ki67 expression at surgery rather than at baseline appears to be a better predictor for disease relapse. © 2001 Cancer Research Campaign  http://www.bjcancer.com PMID:11710821

  5. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same time,…

  6. Stepparents' Affinity-Seeking and Affinity-Maintaining Strategies with Stepchildren.

    ERIC Educational Resources Information Center

    Ganong, Lawrence; Coleman, Marilyn; Fine, Mark; Martin, Patricia

    1999-01-01

    Examines the strategies that stepparents use to develop and maintain affinity with stepchildren and the effects that these strategies have on the development of stepparent-stepchildren relationships. Thirty-one affinity-seeking strategies are identified. Results show that dyadic activities worked best, but it is important that stepchildren…

  7. Sik-ki-mi. Indian Culture Series: Stories of the Blackfeet.

    ERIC Educational Resources Information Center

    Roop, Peter

    The children's story is one of a series about the Blackfeet Tribe at the height of its power in Southern Alberta and North Central Montana. In the story, Eagle Head, a Blackfeet boy, proves his bravery as he faces the first steamboat on the Yellowstone River and recaptures his chief's favorite buffalo horse, Sik-ki-mi, in a raid on a Crow camp.…

  8. Uterine adenosarcomas: diagnostic use of the proliferation marker Ki-67 as an adjunct to morphologic diagnosis.

    PubMed

    Aggarwal, Nidhi; Bhargava, Rohit; Elishaev, Esther

    2012-09-01

    Adenosarcoma (AS) is a rare biphasic neoplasm of the female genital tract characterized by an admixture of benign glandular and low-grade mesenchymal components. The classic low-power growth pattern is periglandular stromal proliferation accompanied by a variable degree of cytologic atypia and mitotic activity. However, as cytologic atypia is an objective criterion, and the number of mitotic figures required for diagnosis is inversely proportional to stromal cytologic atypia, there is a relatively wide variation in the decisive factors used among pathologists to diagnose an AS. Furthermore, the exact number of microscopic fields sufficient for diagnosis and/or the size of the fields adjusted to a specific microscope are not well established. These uncertainties are still an occasional source of misclassification of AS. Our study was conducted to explore the role of immunohistochemistry in the diagnosis of AS. Eight ASs were retrieved and compared with 14 endometrial polyps and 14 atypical polypoid adenomyomas. Immunohistochemical stains for Ki-67, caldesmon, smooth muscle actin, desmin, and CD10 were performed on all cases. All AS had a polypoid growth pattern with a variable increase in periglandular stromal cellularity and stromal nuclear atypia. The mitotic activity ranged from 1 to 15/10 high-power fields, and all AS demonstrated a distinct increase in Ki-67-positive nuclei in the periglandular zone compared with the adjacent stroma, regardless of the mitotic count. The Ki-67-labeling index in periglandular zones was ∼20% compared with <5% in the adjacent stroma. This zonation was not observed in any case of atypical polypoid adenomyomas or endometrial polyps. None of the other stains (CD10, smooth muscle actin, desmin, and caldesmon) helped to differentiate between these entities. Thus, characteristic zonation by Ki-67-staining pattern is a helpful adjunct to the routine morphology in the diagnosis of AS, particularly in curettage specimens, which may lack

  9. KI and WU polyomaviruses and CD4+ cell counts in HIV-1-infected patients, Italy.

    PubMed

    Babakir-Mina, Muhammed; Ciccozzi, Massimo; Farchi, Francesca; Bergallo, Massimiliano; Cavallo, Rossana; Adorno, Gaspare; Perno, Carlo Federico; Ciotti, Marco

    2010-09-01

    To investigate an association between KI and WU polyomavirus (KIPyV and WUPyV) infections and CD4+ cell counts, we tested HIV-1-positive patients and blood donors. No association was found between cell counts and virus infections in HIV-1-positive patients. Frequency of KIPyV infection was similar for both groups. WUPyV was more frequent in HIV-1-positive patients.

  10. KI-catalyzed α-acyloxylation of acetone with carboxylic acids.

    PubMed

    Wu, Ya-Dong; Huang, Bei; Zhang, Yue-Xin; Wang, Xiao-Xu; Dai, Jian-Jun; Xu, Jun; Xu, Hua-Jian

    2016-07-01

    The KI-catalyzed reaction of acetone with aromatic carboxylic acids is achieved, leading to α-acyloxycarbonyl compounds in good to excellent yields under mild reaction conditions. The present method exhibits good functional-group compatibility. Notably, this reaction system is even suitable for cinnamic acid, 3-phenylpropiolic acid and 4-phenylbutanoic acid. A kinetic isotope effect (KIE) study indicates that C-H cleavage of the acetone is the rate-limiting step in the catalytic cycle. PMID:27251323

  11. Activation of c-Ki-ras gene in human pancreatic cancer.

    PubMed

    Prassolov, V S; Sakamoto, H; Nishimura, S; Terada, M; Sugimura, T

    1985-09-01

    DNA isolated from a lymph node with metastasis from pancreatic adenocarcinoma in a Japanese male patient transformed NIH3T3 cells upon transfection by the calcium-phosphate precipitation technique. Analysis of DNA from the transformant revealed the presence of an activated human c-Ki-ras gene, which is considered to be responsible for the transformation of the NIH3T3 cells.

  12. Effectiveness of the KiVa Antibullying Program: Grades 1-3 and 7-9

    ERIC Educational Resources Information Center

    Karna, Antti; Voeten, Marinus; Little, Todd D.; Alanen, Erkki; Poskiparta, Elisa; Salmivalli, Christina

    2013-01-01

    This study investigated the effectiveness of the KiVa Antibullying Program in two samples of students, one from Grades 1-3 (7-9 years old, N = 6,927) and the other from Grades 7-9 (13-15 years old, N = 16, 503). The Grades 1-3 students were located in 74 schools and Grades 7-9 students in 73 schools that were randomly assigned to intervention and…

  13. Affinity enhancement by dendritic side chains in synthetic carbohydrate receptors.

    PubMed

    Destecroix, Harry; Renney, Charles M; Mooibroek, Tiddo J; Carter, Tom S; Stewart, Patrick F N; Crump, Matthew P; Davis, Anthony P

    2015-02-01

    Dendritic side chains have been used to modify the binding environment in anthracene-based synthetic carbohydrate receptors. Control of length, charge, and branching enabled the positioning of side-chain carboxylate groups in such a way that they assisted in binding substrates rather than blocking the cavity. Conformational degeneracy in the dendrimers resulted in effective preorganization despite the flexibility of the system. Strong binding was observed to glucosammonium ions in water, with Ka values up to 7000 M(-1) . Affinities for uncharged substrates (glucose and N-acetylglucosamine) were also enhanced, despite competition from solvent and the absence of electrostatic interactions. PMID:25645064

  14. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    PubMed Central

    Alemán, Fernando; Caballero, Fernando; Ródenas, Reyes; Rivero, Rosa M.; Martínez, Vicente; Rubio, Francisco

    2014-01-01

    Potassium (K+) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter. PMID:25228905

  15. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    PubMed

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  16. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  17. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  18. Twelve cases of Ki-1 positive anaplastic large cell lymphoma of skin.

    PubMed Central

    Banerjee, S S; Heald, J; Harris, M

    1991-01-01

    In seven of 12 cases of Ber-H2 (Ki-1) positive anaplastic large cell non-Hodgkin's lymphoma (Ki-1 ALCL) disease remained localised to skin, and in five there was extracutaneous spread. Four patients had histological evidence of pre-existing or coexisting mycosis fungoides, and three patients had a long standing history of eczema or ichthyosis. In two cases the presence of a T phenotype was shown in frozen sections, and in a further six cases a T phenotype was firmly established in paraffin wax sections. Four patients died less than one year after presentation (two with disseminated lymphoma; two from other causes); one died at five years with widespread lymphoma and the remaining seven cases were alive one to 14 1/2 years after presentation. Three of the four patients with associated mycosis fungoides had prolonged survival, contrary to the findings of previous reports which suggest secondary Ki-1 ALCL behaves aggressively. The recognition of these tumours is important because of their relatively good prognosis. The diagnosis can be readily substantiated immunohistochemically, using a simple panel of antibodies. Images PMID:1650796

  19. Histopathological and Immunohistochemical Evaluation of Meningiomas with Reference to Proliferative Markers p53 and Ki-67

    PubMed Central

    Telugu, Ramesh Babu; Rukmangadha, Nandyala; Patnayak, Rashmi; Phaneendra, Bobbidi Venkata; Prasad, Bodapati Chandra Mowliswara; Reddy, Mandyam Kumaraswamy

    2016-01-01

    Introduction Meningiomas are slow growing primary central nervous system (CNS) tumours attached to the duramater, which arise from the meningothelial cells of the arachnoid. Grading of meningioma based on histological findings assisted with supplementary immunohistochemical studies, predicts the prognosis of meningioma with good precision. Aim To evaluate proliferative markers and correlate with various histological subtypes and grade. Materials and Methods A total of 224 meningiomas, diagnosed between January1995 and October 2011were graded according to WHO 2007 criteria. Immunostaining for p53 and Ki-67 markers were performed on 100 cases. Results There was female predominance. There were 194 Grade I, 24 Grade II and 6 Grade III meningiomas. Brain invasion noted in 18(8%) meningiomas predominantly in grade III followed by grade II. Recurrence was seen in 7 (3.1%) cases, most common in psammomatous followed by angiomatous meningioma. Immunostaining showed p53 positivity in 72.5% of grade I, 83.3% of grade II and all the cases of grade III tumours. Ki-67 Labelling Index (LI) consistently increased from grade I to grade III tumours. Conclusion p53 and Ki-67 LI correlated well with increasing histological grade and biological behaviour of meningioma. PMID:26894073

  20. FEASIBILITY STUDY FOR POTASSIUM IODIDE (KI) DISTRIBUTION IN NEW YORK CITY.

    SciTech Connect

    MOSS, STEVEN

    2005-04-29

    The New York City Department of Health and Mental Hygiene (DOHMH), Bureau of Environmental Science and Engineering, Office of Radiological Health (ORH) [as the primary local technical consultant in the event of a radiological or nuclear incident within the boundaries of New York City] requested the assistance of Brookhaven National Laboratory (BNL) with the development of a Feasibility Study for Potassium Iodide (KI) distribution in the unlikely event of a significant release of radioactive iodine in or near New York City. Brookhaven National Laboratory had previously provided support for New York City with the development of the radiological/nuclear portions of its All Hazards Emergency Response Plans. The work is funded by Medical and Health Research Association (MHRA) of New York City, Inc., under a work grant by the Federal Centers for Disease Control (CDC) for Public Health Preparedness and Response for Bioterrorism. This report is part of the result of that effort. The conclusions of this report are that: (1) There is no credible radiological scenario that would prompt the need for large segments of the general population of New York City to take KI as a result of a projected plume exposure to radioiodine reaching even the lowest threshold of 5 rem to the thyroid; and (2) KI should be stockpiled in amounts and locations sufficient for use by first responders/emergency responders in response to any localized release of radioiodine.

  1. Solvent Controlled Structural Transition of KI4K Self-Assemblies: from Nanotubes to Nanofibrils.

    PubMed

    Zhao, Yurong; Deng, Li; Wang, Jiqian; Xu, Hai; Lu, Jian R

    2015-12-01

    The structural modulation of peptide and protein assemblies under well-controlled conditions is of both fundamental and practical significance. In spite of extensive studies, it remains hugely challenging to tune the self-assembled nanostructures in a controllable manner because the self-assembly processes are dictated by various noncovalent interactions and their interplay. We report here how to manipulate the self-assembly of a designed, symmetric amphiphilic peptide (KI4K) via the solvent-controlled structural transition. Structural transition processes were carefully followed by the combination of transmission electronic microscopy (TEM), atomic force microscopy (AFM), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and small angle neutron scattering (SANS). The results show that the introduction of acetonitrile into water significantly affected the hydrophobic interactions among hydrophobic side chains while imposing little impact on the β-sheet hydrogen bonding between peptide backbones. A structural transition occurred from nanotubes to helical/twisted ribbons and then to thin fibrils with the addition of acetonitrile due to the reduced hydrophobic interactions and the consequent weakening of the lateral stacking between KI4K β-sheets. The increased intermolecular electrostatic repulsions among lysine side chain amino groups had little effect on the lateral stacking of KI4K β-sheets due to the molecular symmetry. Complementary molecular dynamic (MD) simulations also indicated the solvation of acetonitrile molecules into the hydrophobic domains weakening the coherence between the neighboring sheets. PMID:26540520

  2. Self inhibition of phagocytosis: the affinity of ‘Marker of Self’ CD47 for SIRPα dictates potency of inhibition but only at low expression levels

    PubMed Central

    Tsai, Richard K.; Rodriguez, Pia L.; Discher, Dennis E.

    2010-01-01

    Phagocytes engulf foreign cells but not ‘self’ in part because self cells express CD47 as a ligand for signal regulatory protein SIRPα, which inhibits phagocytosis. Motivated by reports of upregulation of CD47 on both normal and cancerous stem cells [1] and also by polymorphisms in SIRPα [2], we show here that inhibition of engulfment correlates with affinity of CD47 for SIRPα – but only at low levels of CD47. One common human polymorph of SIRPα is studied and binds more strongly to human-CD47 than to mouse-CD47 (Kd ≈ 0.12 μM and 6.9 μM, respectively) and does not bind sheep red blood cells (RBC) – which are well-established targets of human macrophages; in comparison, a common mouse polymorph of SIRPα binds with similar affinity to human and mouse CD47 (Kd ≈ 0.22 μM). Using immunoglobulin (IgG)-opsonized particles with varying levels of either human- or mouse-CD47, the effective inhibition constants Ki for blocking phagocytosis are then determined with both human- and mouse-derived macrophages. Only human phagocytes show significant differences in man versus mouse Ki's and only at CD47 levels below normal densities for RBCs. While phospo-signaling through human-SIRPα shows similar trends, consistent again with the affinity differences, saturating levels of CD47 (> Ki) can signal and inhibit phagocytosis regardless of man versus mouse. Quantitative analyses here prompt more complete characterizations of both CD47 levels and SIRPα polymorphisms when attempting to study in vivo effects of these key proteins in innate immunity. PMID:20299253

  3. European and international collaboration in affinity proteomics.

    PubMed

    Stoevesandt, Oda; Taussig, Michael J

    2012-06-15

    In affinity proteomics, specific protein-binding molecules (a.k.a. binders), principally antibodies, are applied as reagents in proteome analysis. In recent years, advances in binder technologies have created the potential for an unprecedented view on protein expression and distribution patterns in plasma, cells and tissues and increasingly on protein function. Particular strengths of affinity proteomics methods include detecting proteins in their natural environments of cell or tissue, high sensitivity and selectivity for detection of low abundance proteins and exploiting binding actions such as functional interference in living cells. To maximise the use and impact of affinity reagents, it will be essential to create comprehensive, standardised binder collections. With this in mind, the EU FP7 programme AFFINOMICS (http://www.affinomics.org), together with the preceding EU programmes ProteomeBinders and AffinityProteome, aims to extend affinity proteomics research by generating a large-scale resource of validated protein-binding molecules for characterisation of the human proteome. Activity is directed at producing binders to about 1000 protein targets, primarily in signal transduction and cancer, by establishing a high throughput, coordinated production pipeline. An important aspect of AFFINOMICS is the development of highly efficient recombinant selection methods, based on phage, cell and ribosome display, capable of producing high quality binders at greater throughput and lower cost than hitherto. The programme also involves development of innovative and sensitive technologies for specific detection of target proteins and their interactions, and deployment of binders in proteomics studies of clinical relevance. The need for such binder generation programmes is now recognised internationally, with parallel initiatives in the USA for cancer (NCI) and transcription factors (NIH) and within the Human Proteome Organisation (HUPO). The papers in this volume of New

  4. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  5. The dynamics of metric-affine gravity

    SciTech Connect

    Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

    2011-05-15

    Highlights: > The role and the dynamics of the connection in metric-affine theories is explored. > The most general second order action does not lead to a dynamical connection. > Including higher order invariants excites new degrees of freedom in the connection. > f(R) actions are also discussed and shown to be a non- representative class. - Abstract: Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy theories to

  6. [Tobacco and alcohol consumption among 11- to 17-year-old adolescents: results of the KiGGS study: first follow-up (KiGGS Wave 1)].

    PubMed

    Lampert, T; Kuntz, B

    2014-07-01

    In this paper, tobacco and alcohol consumption among adolescents in Germany was analyzed. In addition to the current situation, we report temporal developments and trends. Data were obtained from the first follow-up of the KiGGS study (KiGGS Wave 1) conducted from 2009 to 2012. All girls and boys aged 11-17 years (n = 5,258) were included. The results show that currently 12.0% of 11- to 17-year-old adolescents in Germany smoke, 5.4% of them on a daily basis. At-risk drinking (AUDIT-C total score) was prevalent among 15.8% of adolescents, heavy episodic drinking (six or more alcoholic standard drinks on a single occasion at least once a month) among 11.5%. No significant gender differences were found for most indicators. However, among adolescents aged 14-17 years, boys revealed a greater inclination toward heavy episodic drinking than girls did (23.1 vs. 16.5 %, p < 0.01). Regarding smoking, distinct socioeconomic differences were observed. For example, adolescents from families with a low socioeconomic status (SES) smoke significantly more often on a regular or daily basis compared with their peers from high-SES families (OR = 1.95, 95% CI = 1.16-3.27 and OR = 3.71, 95% CI = 2.05-6.69, respectively). The relationship between SES and alcohol consumption is rather weak. Significant differences emerged only regarding lifetime prevalence of alcohol consumption, and indicate lower consumption rates among low-SES compared with high-SES adolescents (OR = 0.47, 95% CI = 0.33-0.68). Consideration of the KiGGS baseline study data (2003-2006) shows that smoking prevalence has dropped almost by half from 20.4 to 12.0%. The percentage of adolescents who have ever drunk alcohol has decreased from 62.8 to 54.4%. These results are consistent with the findings of other studies on adolescent tobacco and alcohol consumption and should be considered in the context of preventive efforts that have been strengthened in recent years, especially regarding

  7. Affine Invariant Character Recognition by Progressive Removing

    NASA Astrophysics Data System (ADS)

    Iwamura, Masakazu; Horimatsu, Akira; Niwa, Ryo; Kise, Koichi; Uchida, Seiichi; Omachi, Shinichiro

    Recognizing characters in scene images suffering from perspective distortion is a challenge. Although there are some methods to overcome this difficulty, they are time-consuming. In this paper, we propose a set of affine invariant features and a new recognition scheme called “progressive removing” that can help reduce the processing time. Progressive removing gradually removes less feasible categories and skew angles by using multiple classifiers. We observed that progressive removing and the use of the affine invariant features reduced the processing time by about 60% in comparison to a trivial one without decreasing the recognition rate.

  8. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  9. Adsorption affinity of anions on metal oxyhydroxides

    NASA Astrophysics Data System (ADS)

    Pechenyuk, S. I.; Semushina, Yu. P.; Kuz'mich, L. F.

    2013-03-01

    The dependences of anion (phosphate, carbonate, sulfate, chromate, oxalate, tartrate, and citrate) adsorption affinity anions from geometric characteristics, acid-base properties, and complex forming ability are generalized. It is shown that adsorption depends on the nature of both the anions and the ionic medium and adsorbent. It is established that anions are generally grouped into the following series of adsorption affinity reduction: PO{4/3-}, CO{3/2-} > C2O{4/2-}, C(OH)(CH2)2(COO){3/3-}, (CHOH)2(COO){2/2-} > CrO{4/2-} ≫ SO{4/2-}.

  10. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  11. How Reliable Is Ki-67 Immunohistochemistry in Grade 2 Breast Carcinomas? A QA Study of the Swiss Working Group of Breast- and Gynecopathologists

    PubMed Central

    Varga, Zsuzsanna; Diebold, Joachim; Dommann-Scherrer, Corina; Frick, Harald; Kaup, Daniela; Noske, Aurelia; Obermann, Ellen; Ohlschlegel, Christian; Padberg, Barbara; Rakozy, Christiane; Sancho Oliver, Sara; Schobinger-Clement, Sylviane; Schreiber-Facklam, Heide; Singer, Gad; Tapia, Coya; Wagner, Urs; Mastropasqua, Mauro G.; Viale, Giuseppe; Lehr, Hans-Anton

    2012-01-01

    Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas. PMID:22662150

  12. 8-NH2-boldine, an antagonist of alpha1A and alpha1B adrenoceptors without affinity for the alpha1D subtype: structural requirements for aporphines at alpha1-adrenoceptor subtypes.

    PubMed

    Ivorra, M Dolores; Valiente, Miguel; Martínez, Sonia; Madrero, Yolanda; Noguera, M Antonia; Cassels, Bruce K; Sobarzo, Eduardo M; D'Ocon, Pilar

    2005-10-01

    Structure-activity analysis of 21 aporphine derivatives was performed by examining their affinities for cloned human alpha (1A), alpha (1B) and alpha (1D) adrenoceptors (AR) using membranes prepared from rat-1 fibroblasts stably expressing each alpha (1)-AR subtype. All the compounds tested competed for [ (125)I]-HEAT binding with steep and monophasic curves. The most interesting compound was 8-NH (2)-boldine, which retains the selective affinity for alpha(1A)-AR (pKi = 6.37 +/- 0.21) vs. alpha(1B)-AR (pKi = 5.53 +/- 0.11) exhibited by 1,2,9,10-tetraoxygenated aporphines, but shows low affinity for alpha(1D)-AR (pKi < 2.5). Binding studies on native adrenoceptors present in rat cerebral cortex confirms the results obtained for human cloned alpha (1)-AR subtypes. The compounds selective for the alpha (1A) subtype discriminate two binding sites in rat cerebral cortex confirming a mixed population of alpha (1A)- and alpha (1B)-AR in this tissue. All compounds are more selective as inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. A close relationship was found between affinities obtained for cloned alpha (1A)-AR and inhibitory potencies on noradrenaline-induced contraction or inositol phosphate accumulation in tail artery, confirming that there is a homogeneous functional population of alpha(1A)-AR in this vessel. On the contrary, a poor correlation seems to exist between the affinity of 8-NH (2)-boldine for cloned alpha (1D)-AR and its potency as an inhibitor of noradrenaline-induced contraction or inositol phosphate accumulation in rat aorta, which confirms that a heterogeneous population of alpha (1)-AR mediates the adrenergic response in this vessel.

  13. KiSS-1-mediated suppression of the invasive ability of human pancreatic carcinoma cells is not dependent on the level of KiSS-1 receptor GPR54

    PubMed Central

    WANG, CHUN-HUI; QIAO, CHONG; WANG, RUO-CHEN; ZHOU, WEN-PING

    2016-01-01

    The onset of local invasion and lymphatic metastasis in pancreatic cancer limits survival following surgical intervention and additional therapies. Reduced expression of KiSS-1 in pancreatic cancer is associated with cancer metastasis. Previous studies have indicated that kisspeptin, the KiSS-1 peptide, is able to bind to its receptor-GPR54 (hOT7T175) and suppress the migration of PANC-1 pancreatic cancer cells. Whether the metastatic suppression of KiSS-1 is dependent on the levels of GPR54 in pancreatic cancer cell lines remains unclear. Human BxPC-3 pancreatic carcinoma cells are highly differentiated without exhibiting metastasis, however PANC-1 pancreatic carcinoma cells are poorly differentiated and exhibit local and lymph node metastasis. Compared with primary cultured trophoblasts, BxPc-3 and PANC-1 cells were observed to express low levels of KiSS-1 mRNA and protein, measured using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. However, greater mRNA and protein expression levels of GPR54 were observed in PANC-1 cells compared with BxPc-3 cells. An MTT assay was used to investigate the effect of KiSS-1 on BxPc-3 and PANC-1 cell proliferation. There were no significant differences in proliferation following transfection with KiSS-1 in BxPc-3 and PANC-1 cells compared with the controls (P>0.05). A Transwell assay with chambers coated with Matrigel was used to evaluate the in vitro invasive ability of BxPc-3 and PANC-1 cells, with the invasion index of BxPc-3 and PANC-1 cells significantly reduced following 48 h of KiSS-1 overexpression (P<0.05). The mRNA and protein expression levels of KiSS-1 were significantly increased in BxPc-3 and PANC-1 cells 48 h subsequent to transfection with KiSS-1 (P<0.05), while GPR54 expression was not altered (P>0.05). KiSS-1 is a metastasis suppressor gene of pancreatic cancer, and this suppression is not dependent on the expression levels of GPR54. Therefore, KiSS-1 is

  14. Plasma Asp13-Ki-ras oncoprotein expression in vinyl chloride monomer workers in Taiwan.

    PubMed

    Luo, J C; Liu, H T; Cheng, T J; Du, C L; Wang, J D

    1998-12-01

    Vinyl chloride (VC) workers are known to be at risk for development of liver angiosarcoma, a rare tumor. Previously, more than 80% of VC workers with liver angiosarcoma have been found to have an Asp-13 c-Ki-ras oncogene mutation, and more than 50% of VC-exposed workers without liver tumors were found to have Asp13-Ki-ras oncoprotein in their plasma. Some workers in Taiwan had also been exposed to VC, and some have contracted liver tumors. In this study, we used enhanced chemiluminescence Western blotting to detect Asp13-p21-Ki-ras in the sera of VC-exposed workers in Taiwan. There were 14 of 113 (12.4%) VC workers positive for the Asp13-Ki-ras oncoprotein in plasma, but 0 of 18 controls were positive. There were 10 of 69 (14.5%) plasma-positives among the more highly exposed (> 1000 ppm-months) workers and 4 of 48 (9.1%) plasma-positives among the lesser exposed (< or = 1000 ppm-months). Compared with the unexposed controls, the odds ratios (and 95% confidence intervals [CI]) for plasma-positivity were 4.11 (95% CI = 0.21, 80.4) in the lower-exposed workers and 6.53 (95% CI = 0.37, 116.9) in the higher-exposed workers, and there was a linear trend between exposure and plasma-positivity (P = 0.073). After adjusting for age and drinking status, the odds ratios (and 95% CIs) were 1.64 (95% CI = 0.17, 15.8), and 2.65 (95% CI = 0.42, 16.8), respectively, and there was a significant linear trend between exposure and plasma-positivity (P = 0.048). In summary, Asp13-Ki-ras oncoprotein can be found in the plasma of VC workers in Taiwan, and a significant dose-response relationship exists between plasma oncoprotein expression and VC exposure.

  15. Tracking EcoKI and DNA fifty years on: a golden story full of surprises.

    PubMed

    Loenen, Wil A M

    2003-12-15

    1953 was a historical year for biology, as it marked the birth of the DNA helix, but also a report by Bertani and Weigle on 'a barrier to infection' of bacteriophage lambda in its natural host, Escherichia coli K-12, that could be lifted by 'host-controlled variation' of the virus. This paper lay dormant till Nobel laureate Arber and PhD student Dussoix showed that the lambda DNA was rejected and degraded upon infection of different bacterial hosts, unless it carried host-specific modification of that DNA, thus laying the foundations for the phenomenon of restriction and modification (R-M). The restriction enzyme of E.coli K-12, EcoKI, was purified in 1968 and required S-adenosylmethionine (AdoMet) and ATP as cofactors. By the end of the decade there was substantial evidence for a chromosomal locus hsdK with three genes encoding restriction (R), modification (M) and specificity (S) subunits that assembled into a large complex of >400 kDa. The 1970s brought the message that EcoKI cut away from its DNA recognition target, to which site the enzyme remained bound while translocating the DNA past itself, with concomitant ATP hydrolysis and subsequent double-strand nicks. This translocation event created clearly visible DNA loops in the electron microscope. EcoKI became the archetypal Type I R-M enzyme with curious DNA translocating properties reminiscent of helicases, recognizing the bipartite asymmetric site AAC(N6)GTGC. Cloning of the hsdK locus in 1976 facilitated molecular understanding of this sophisticated R-M complex and in an elegant 'pas de deux' Murray and Dryden constructed the present model based on a large body of experimental data plus bioinformatics. This review celebrates the golden anniversary of EcoKI and ends with the exciting progress on the vital issue of restriction alleviation after DNA damage, also first reported in 1953, which involves intricate control of R subunit activity by the bacterial proteasome ClpXP, important results that will keep

  16. [Physical activity and electronic media use in children and adolescents: results of the KiGGS study: first follow-up (KiGGS wave 1)].

    PubMed

    Manz, K; Schlack, R; Poethko-Müller, C; Mensink, G; Finger, J; Lampert, T

    2014-07-01

    Physical activity during childhood and adolescence has numerous health benefits, while sedentary behavior, especially electronic media use, is associated with the development of overweight. Therefore, the promotion of physical activity during childhood and adolescence is an integral part of national public health efforts. The aim of this article is to describe the physical activity behavior of German children and adolescents based on the nationwide data of the German Health Interview and Examination Survey for Children and Adolescents (KiGGS wave 1). Furthermore, the association between physical activity and sports participation and use of screen-based media in youth aged 11 to 17 years was analyzed. The analyses included data from 10,426 children and adolescents aged 3-17 years collected by telephone interviews. Children older than 11 years answered the questions by themselves, whereas a parent was interviewed for younger children. The descriptive analyses were performed under consideration of social and demographic factors. According to the results of KiGGS wave 1 a total of 77.5% (95% Cl 76.0-78.9 %) of the children and adolescents participated in sports activities, and 59.7% (58.1-61.3 %) were members of a sports club. The recommendation of the World Health Organization (WHO) to be physically active at least 60 min per day was achieved by 27.5% (26.0-28.9 %). Children and adolescents with a low socioeconomic status (SES) participated less in sports activities than children of higher SES groups. Excessive use of screen-based media was more likely to be associated with lack of sports participation than with a lack of physical activity. In the future, preventive measures should promote the daily physical activity of children and adolescents and additionally encourage children and adolescents with low SES to participate in sports activities. PMID:24950833

  17. [Physical activity and electronic media use in children and adolescents: results of the KiGGS study: first follow-up (KiGGS wave 1)].

    PubMed

    Manz, K; Schlack, R; Poethko-Müller, C; Mensink, G; Finger, J; Lampert, T

    2014-07-01

    Physical activity during childhood and adolescence has numerous health benefits, while sedentary behavior, especially electronic media use, is associated with the development of overweight. Therefore, the promotion of physical activity during childhood and adolescence is an integral part of national public health efforts. The aim of this article is to describe the physical activity behavior of German children and adolescents based on the nationwide data of the German Health Interview and Examination Survey for Children and Adolescents (KiGGS wave 1). Furthermore, the association between physical activity and sports participation and use of screen-based media in youth aged 11 to 17 years was analyzed. The analyses included data from 10,426 children and adolescents aged 3-17 years collected by telephone interviews. Children older than 11 years answered the questions by themselves, whereas a parent was interviewed for younger children. The descriptive analyses were performed under consideration of social and demographic factors. According to the results of KiGGS wave 1 a total of 77.5% (95% Cl 76.0-78.9 %) of the children and adolescents participated in sports activities, and 59.7% (58.1-61.3 %) were members of a sports club. The recommendation of the World Health Organization (WHO) to be physically active at least 60 min per day was achieved by 27.5% (26.0-28.9 %). Children and adolescents with a low socioeconomic status (SES) participated less in sports activities than children of higher SES groups. Excessive use of screen-based media was more likely to be associated with lack of sports participation than with a lack of physical activity. In the future, preventive measures should promote the daily physical activity of children and adolescents and additionally encourage children and adolescents with low SES to participate in sports activities.

  18. [Health-related quality of life in children and adolescents in Germany: results of the KiGGS study: first follow-up (KiGGS Wave 1)].

    PubMed

    Ellert, U; Brettschneider, A-K; Ravens-Sieberer, U

    2014-07-01

    In recent years, there has been a change in the health and disease spectrum among children and adolescents, with an increase in mental health problems and a shift from acute to chronic illness. In this phase, the health-related quality of life (HRQoL) has increased in importance as a dimension of subjective health. The aim of this study is to describe the HRQoL of children and adolescents measured with the internationally standardized screening instrument KIDSCREEN-10. In the follow-up of the KiGGS study in 2009-2012 (KiGGS Wave 1), 2,567 parents of children aged 7-10 years and 4,878 adolescents aged 11 years or older completed the KIDSCREEN-10 questionnaire. In all, 94% of parents of 7- to 10-year-old girls and boys estimate the HRQoL of their children to be "very good" or "good." Of the 11- to 17-year-old adolescents, 96% report their HRQoL as "very good" or "good." Somatic diseases and pain as well as mental health problems and a low social status are included in the HRQoL in only a limited way. Potential differences in HRQoL by social status were not confirmed in multivariate models. The HRQoL of the examined children and adolescents is predominantly very good or good. Interventions to improve the HRQoL of children and adolescents with diseases and psychopathological problems are necessary, regardless of their social status.

  19. Potassium Iodide ("KI"): Instructions to Make Potassium Iodide Solution for Use During a Nuclear Emergency (Liquid Form)

    MedlinePlus

    ... make Potassium Iodide Solution for Use During a Nuclear Emergency (Liquid Form) Share Tweet Linkedin Pin it ... Preparation and Dosing Instructions for Use During a Nuclear Emergency To Make KI Solution (Liquid Form), using ...

  20. Interpolation method for accurate affinity ranking of arrayed ligand-analyte interactions.

    PubMed

    Schasfoort, Richard B M; Andree, Kiki C; van der Velde, Niels; van der Kooi, Alex; Stojanović, Ivan; Terstappen, Leon W M M

    2016-05-01

    The values of the affinity constants (kd, ka, and KD) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (kd and ka) and dissociation equilibrium (KD) constants for various ligand densities and analyte concentrations are exponentially interpolated to the KD at Rmax = 100 RU response level (KD(R100)).

  1. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  2. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  3. Two bradykinin binding sites with picomolar affinities

    SciTech Connect

    Manning, D.C.; Vavrek, R.; Stewart, J.M.; Snyder, S.H.

    1986-05-01

    Bradykinin (BK) and related peptides exert a wide range of effects on several organ systems. We have attempted to sort out these effects by studying the binding interaction of (/sup 3/H)BK at the membrane level with in vitro receptor binding techniques. High specific activity (/sup 3/H)BK and an enzyme inhibitor cocktail has enabled us to label two BK binding sites with different affinity and peptide specificity in several guinea-pig tissues. In the guinea-pig ileum the high-affinity site has an equilibrium dissociation constant (Kd) for (/sup 3/H)BK of 13 pM and a maximal number of binding sites of 8.3 pmol/g of tissue wet weight. The low-affinity guinea-pig ileum site displays a Kd of 910 pM, a maximum number of binding sites of 14 pmol/g of tissue wet weight and shows a greater selectivity for BK analogs over Lysyl-BK analogs. Two similar sites can also be discriminated in kidney and heart. The potencies of a series of BK analogs at the high-affinity guinea-pig ileum site correlate well with their potencies in contracting ileal smooth muscle. The binding of (/sup 3/H)BK in the guinea-pig ileum is inhibited by physiological concentrations of monovalent and divalent cations.

  4. Ki-67 is overexpressed in human laryngeal carcinoma and contributes to the proliferation of HEp2 cells

    PubMed Central

    Bai, Yanxia; Shao, Yuan; Li, Huajing; Xue, Wanli; Quan, Fang; Wu, Shengli

    2016-01-01

    Ki-67 is one of the most useful markers to evaluate cell proliferative activity and has been widely used in tumor treatment and research. However, its role in human laryngeal carcinoma remains poorly defined. The aim of the present study was to investigate the expression of Ki-67 in human laryngeal squamous carcinoma and the effect of Ki-67 gene silencing by small interfering (si)RNA on the proliferation of human laryngocarcinoma HEp2 cells. Immunohistochemistry and reverse transcription-quantitative polymerase chain reaction were performed to examine the expression of Ki-67 in human laryngeal squamous carcinoma tissues and adjacent non-cancer tissues from 50 patients with laryngeal squamous carcinoma. RNA interference was used to knock down the expression of Ki-67 in the HEp2 cell line, and the proliferation of the treated cells was observed in vitro. Western blot analysis was used to determine the expression levels of epidermal growth factor receptor (EGFR) and E-cadherin in the treated cells. The expression of Ki-67 in the laryngeal squamous carcinoma tissues was significantly higher than that of the adjacent non-tumor tissues (P=0.028). The high expression of Ki-67 in cancer was significantly correlated with cervical lymph node metastasis and clinical outcomes (all P<0.001). The silencing of Ki-67 resulted in the inhibition of proliferation of the HEp2 human laryngocarcinoma cells (P<0.001). In addition, compared with the control group, the expression levels of EGFR and E-cadherin in the Ki-67 siRNA-treated cells were significantly decreased (P<0.001) and increased (P<0.001), respectively. These results suggested that Ki-67 is important in regulating the proliferation of human laryngocarcinoma HEp2 cells and that the mechanism may at least partially be associated with the upregulation of EGFR and the downregulation of E-cadherin. Overall, Ki-67 can be used as an important indicator for judging clinical progress and estimating prognosis in human laryngeal

  5. St Gallen 2015 subtyping of luminal breast cancers: impact of different Ki67-based proliferation assessment methods.

    PubMed

    Focke, Cornelia M; van Diest, Paul J; Decker, Thomas

    2016-09-01

    Ki67 has been proposed as prognostic proliferation marker in luminal breast cancer (BC), but little is known on the influence of Ki67 assessment methods on subtyping into luminal A- and B-like tumors. Our aim was to study the influence of different Ki67-labeling index (Ki67-LI) assessment methods on the proportion of BCs classified as luminal A-like. 280 early BCs were subtyped according to the St Gallen 2015 definitions into 71 % luminal (HER2 negative), 6 % luminal B-like (HER2 positive), 13 % triple negative, 1 % HER2 positive (nonluminal), and 9 % special type. Digitized whole slides were counted manually on the screen. We used nine defined counting methods to assess the Ki67-LI (including the International Ki67 in Breast Cancer Working Group recommendations), and compared the resulting medians and the proportions of cancers classified as luminal A-like according to the formerly used cut-off <20 %. Methods assessing hot spots and tumor periphery resulted in significantly higher Ki67-LI medians than those measuring an average proliferation (27.45 % vs 16.96 %, p < 0.0001). Substantially lower median Ki67-LI were found when assessing 1020 compared to counting 100, 200, 300 cells (17.65 vs 33 %, vs 28 %, vs 24.33 %, respectively; p < 0.0001), or 510 cells (20.59 %, p = 0.019). Applying a standard Ki67-LI cut-off <20 % to define low proliferation for all methods, the proportion of luminal A-like cancers varied between 13 and 44 %. The proportion of BCs classified as luminal A-like is highly influenced by the Ki67-LI assessment method. As a consequence, the selection of a specific Ki67-LI assessment method may have a direct effect on the proportion of patients considered having low-risk disease and thus influence therapeutic decision making. This calls for a standardized assessment method. PMID:27558625

  6. Somatostatin analogues according to Ki67 index in neuroendocrine tumours: an observational retrospective-prospective analysis from real life.

    PubMed

    Faggiano, Antongiulio; Carratù, Anna Chiara; Guadagno, Elia; Tafuto, Salvatore; Tatangelo, Fabiana; Riccardi, Ferdinando; Mocerino, Carmela; Palmieri, Giovannella; Damiano, Vincenzo; Siciliano, Roberta; Leo, Silvana; Mauro, Annamaria; Tozzi, Lucia Franca; Battista, Claudia; De Rosa, Gaetano; Colao, Annamaria

    2016-02-01

    Somatostatin analogues (SSAs) have shown limited and variable antiproliferative effects in neuroendocrine tumours (NETs). Whether tumour control by SSAs depends on grading based on the 2010 WHO NET classification is still unclear. The aim of this study is to evaluate the efficacy of long-acting SSAs in NETs according to Ki67 index. An observational Italian multicentre study was designed to collect data in patients with gastro-entero-pancreatic or thoracic NETs under SSA treatment. Both retrospective and prospective data were included and they were analysed in line with Ki67 index, immunohistochemically evaluated in tumour samples and graded according to WHO classification (G1 = Ki67 index 0-2%, G2 = Ki67 index 3-20%, G3 = Ki67 index > 20%). Among 601 patients with NET, 140 with a histologically confirmed gastro-entero-pancreatic or thoracic NET or NET with unknown primary were treated with lanreotide autogel or octreotide LAR. An objective tumour response was observed in 11%, stability in 58% and progression in 31%. Objective response and tumour stability were not significantly different between G1 and G2 NETs. Progression free survival was longer but not significantly different in G1 than G2 NETs (median: 89 vs 43 months, p = 0.15). The median PFS was significantly longer in NETs showing Ki67 < 5% than in those showing Ki67 ≥ 5% (89 vs 35 months, p = 0.005). SSA therapy shows significant antiproliferative effects in well differentiated low/intermediate-proliferating NETs, not only G1 but also in G2 type. A Ki67 index of 5% seems to work better than 3% to select the best candidates for SSA therapy. PMID:26701729

  7. An analysis of potassium iodide (KI) prophylaxis for the general public in the event of a nuclear accident

    SciTech Connect

    Behling, H.; Behling, K.; Amarasooriya, H.; Kotsch, J.

    1995-02-01

    A generic difficulty encountered in cost-benefit analyses is the quantification of major elements that define the costs and the benefits in commensurate units. In this study, the costs of making KI available for public use, and the avoidance of thyroidal health effects predicted to be realized from the availability of that KI (i.e., the benefits), are defined in the commensurate units of dollars.

  8. Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation

    PubMed Central

    Capoferri, Luigi; Verkade-Vreeker, Marlies C. A.; Buitenhuis, Danny; Commandeur, Jan N. M.; Pastor, Manuel; Vermeulen, Nico P. E.; Geerke, Daan P.

    2015-01-01

    Prediction of human Cytochrome P450 (CYP) binding affinities of small ligands, i.e., substrates and inhibitors, represents an important task for predicting drug-drug interactions. A quantitative assessment of the ligand binding affinity towards different CYPs can provide an estimate of inhibitory activity or an indication of isoforms prone to interact with the substrate of inhibitors. However, the accuracy of global quantitative models for CYP substrate binding or inhibition based on traditional molecular descriptors can be limited, because of the lack of information on the structure and flexibility of the catalytic site of CYPs. Here we describe the application of a method that combines protein-ligand docking, Molecular Dynamics (MD) simulations and Linear Interaction Energy (LIE) theory, to allow for quantitative CYP affinity prediction. Using this combined approach, a LIE model for human CYP 1A2 was developed and evaluated, based on a structurally diverse dataset for which the estimated experimental uncertainty was 3.3 kJ mol-1. For the computed CYP 1A2 binding affinities, the model showed a root mean square error (RMSE) of 4.1 kJ mol-1 and a standard error in prediction (SDEP) in cross-validation of 4.3 kJ mol-1. A novel approach that includes information on both structural ligand description and protein-ligand interaction was developed for estimating the reliability of predictions, and was able to identify compounds from an external test set with a SDEP for the predicted affinities of 4.6 kJ mol-1 (corresponding to 0.8 pKi units). PMID:26551865

  9. Antibody response and antibody affinity maturation in cats with experimental proliferative immune complex glomerulonephritis.

    PubMed

    Bishop, S A; Bailey, M; Lucke, V M; Stokes, C R

    1992-07-01

    An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an

  10. Effect of KiFAY on Performance, Insulin-like Growth Factor-1, and Thyroid Hormones in Broilers

    PubMed Central

    Kini, Amit; Fernandes, Custan; Suryawanshi, Dayaram

    2016-01-01

    A comparative study was performed to investigate the efficacy of KiFAY as a feed additive on performance parameters, thyroid, and pancreatic hormone levels in broilers. Ninety birds (Vencobb 400) were randomly divided into three groups viz., Control (no DL-methionine supplementation), Treatment1 (containing added DL-methionine) and Treatment 2 (containing KiFAY and without DL-methionine supplementation). The performance parameters (weekly body weight, body weight gain, feed intake, and feed consumption ratio) were recorded and calculated during the whole study of 4 weeks. Analyses of insulin and insulin-like growth factor (IGF 1), triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) were performed at the end of the study. The results show that birds on supplementation of KiFAY performed significantly (p<0.001) better than other treatments. The weekly body weight, body weight gain, feed in-take and feed consumption ratio improved in KiFAY treated birds. The study found an increase in insulin and IGF1 levels (p<0.001) in KiFAY compared with the other treatments. Serum T3, T4, and TSH levels in the Treatment 2 were higher than other treatments (p<0.001). The KiFAY supplementation was able to improve performance with associated responses at a hormonal level in broilers. PMID:27221245

  11. Methyl cation affinities of neutral and anionic maingroup-element hydrides: trends across the periodic table and correlation with proton affinities.

    PubMed

    Mulder, R Joshua; Guerra, Célia Fonseca; Bickelhaupt, F Matthias

    2010-07-22

    We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and corresponding entropies) at 298 K of all anionic (XH(n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (i.e., group 18) along the periods 2-6. The cation affinity of the bases decreases from H(+) to CH(3)(+). To understand this trend, we have carried out quantitative bond energy decomposition analyses (EDA). Quantitative correlations are established between the MCA and PA values.

  12. Methyl cation affinities of neutral and anionic maingroup-element hydrides: trends across the periodic table and correlation with proton affinities.

    PubMed

    Mulder, R Joshua; Guerra, Célia Fonseca; Bickelhaupt, F Matthias

    2010-07-22

    We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and corresponding entropies) at 298 K of all anionic (XH(n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (i.e., group 18) along the periods 2-6. The cation affinity of the bases decreases from H(+) to CH(3)(+). To understand this trend, we have carried out quantitative bond energy decomposition analyses (EDA). Quantitative correlations are established between the MCA and PA values. PMID:20575582

  13. What Value "Value Added"?

    ERIC Educational Resources Information Center

    Richards, Andrew

    2015-01-01

    Two quantitative measures of school performance are currently used, the average points score (APS) at Key Stage 2 and value-added (VA), which measures the rate of academic improvement between Key Stage 1 and 2. These figures are used by parents and the Office for Standards in Education to make judgements and comparisons. However, simple…

  14. KiSS-1 in the mammalian ovary: distribution of kisspeptin in human and marmoset and alterations in KiSS-1 mRNA levels in a rat model of ovulatory dysfunction.

    PubMed

    Gaytán, F; Gaytán, M; Castellano, J M; Romero, M; Roa, J; Aparicio, B; Garrido, N; Sánchez-Criado, J E; Millar, R P; Pellicer, A; Fraser, H M; Tena-Sempere, M

    2009-03-01

    Kisspeptins, the products of the KiSS-1 gene acting via G protein-coupled receptor 54 (GPR54), have recently emerged as pivotal signals in the hypothalamic network triggering the preovulatory surge of gonadotropins and, hence, ovulation. Additional actions of kisspeptins at other levels of the hypothalamic-pituitary-ovarian axis have been suggested but remain to date scarcely studied. We report herein the pattern of expression of KiSS-1 and GPR54 in the human and nonhuman primate ovary and evaluate changes in ovarian KiSS-1 expression in a rat model of ovulatory dysfunction. KiSS-1 and GPR54 mRNAs were detected in human ovarian tissue and cultured granulosa-lutein cells. In good agreement, kisspeptin immunoreactivity was observed in cyclic human and marmoset ovaries, with prominent signals in the theca layer of growing follicles, corpora lutea, interstitial gland, and ovarian surface epithelium. GPR54 immunoreactivity was also found in human theca and luteal cells. Administration of indomethacin to cyclic female rats disturbed ovulation and resulted in a dramatic drop in ovarian KiSS-1, but not GPR54, cyclooxygenase-2 (COX-2), or progesterone receptor, mRNA levels at the time of ovulation; an effect mimicked by the selective COX-2 inhibitor NS398 and rescued by coadministration of PGE(2). Likewise, the stimulatory effect of human choriogonadotropin on ovarian KiSS-1 expression was partially blunted by indomethacin. In contrast, KiSS-1 mRNA levels remained unaltered in another model of ovulatory failure, i.e., the RU486-treated rat. In summary, we document for the first time the expression of KiSS-1/kisspeptin and GPR54 in the human and nonhuman primate ovary. In addition, we provide evidence for the ability of inhibitors of COX-2, known to disturb follicular rupture and ovulation, to selectively alter the expression of KiSS-1 gene in rat ovary. Altogether, our results are suggestive of a conserved role of local KiSS-1 in the direct control of ovarian functions in

  15. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  16. Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments.

    PubMed

    Della Ducata, Daniela; Jaehrling, Jan; Hänel, Cornelia; Satzger, Marion; Wolber, Meike; Ostendorp, Ralf; Pabst, Stefan; Brocks, Bodo

    2015-12-01

    The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the binding partners are not labeled, the resulting KD represents a sound approximation of the real affinity. For screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of a soluble target molecule, and unbound antibodies are subsequently quantified on 384-well Meso Scale Discovery (MSD) plates coated with the respective antigen. For determination of KD values from the resulting titration curves, fit models deduced from the law of mass action for 1:1 and 2:1 binding modes are applied to assess hundreds of interactions simultaneously. The accuracy of the method is demonstrated by comparing results from different screening campaigns from affinity optimization projects with results from detailed affinity characterization.

  17. Synthesis and structure-affinity relationships of new 4-(6-iodo-H-imidazo[1,2-a]pyridin-2-yl)-N-dimethylbenzeneamine derivatives as ligands for human beta-amyloid plaques.

    PubMed

    Cai, Lisheng; Cuevas, Jessica; Temme, Sebastian; Herman, Mary M; Dagostin, Claudio; Widdowson, David A; Innis, Robert B; Pike, Victor W

    2007-09-20

    A new and extensive set of 4-(6-iodo-H-imidazo[1,2-a]pyridin-2-yl)-N-dimethylbenzeneamine (IMPY) derivatives was synthesized and assayed for affinity toward human Abeta plaques. 6-Ethylthio- (12h), 6-cyano- (12e), 6-nitro- (12f), and 6-p-methoxybenzylthio- (15d) analogues were discovered to have high affinity (KI < 10 nM). However, introduction of a hydrophilic thioether group in the 6-position (15a-c, 15e-g) reduced or abolished affinity. In secondary N-methyl analogues, a bromo substituent in the adjacent ring position (14a) imparted high affinity (KI = 7.4 nM) whereas a methyl substituent did not (14c). The tolerance for nonhydrophilic thioether substituents in the 6-position opens up the possibility of developing new sensitive positron emission tomography radioligands for imaging human Abeta plaques in Alzheimer's disease, especially in view of the amenability of thioethers to be labeled with carbon-11 or fluorine-18 through S-alkylation reactions. The structure-activity relationships revealed in this study extends insight into the topography of the binding site for IMPY-like ligands in human Abeta plaques. PMID:17722900

  18. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity.

    PubMed Central

    Rajas, F; Rousset, B

    1993-01-01

    We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme. Images Figure 1 Figure 3

  19. Novel Kac-Moody-type affine extensions of non-crystallographic Coxeter groups

    NASA Astrophysics Data System (ADS)

    Dechant, Pierre-Philippe; Bœhm, Céline; Twarock, Reidun

    2012-07-01

    Motivated by recent results in mathematical virology, we present novel asymmetric {Z}[\\tau ]-integer-valued affine extensions of the non-crystallographic Coxeter groups H2, H3 and H4 derived in a Kac-Moody-type formalism. In particular, we show that the affine reflection planes which extend the Coxeter group H3 generate (twist) translations along two-, three- and five-fold axes of icosahedral symmetry, and we classify these translations in terms of the Fibonacci recursion relation applied to different start values. We thus provide an explanation of previous results concerning affine extensions of icosahedral symmetry in a Coxeter group context, and extend this analysis to the case of the non-crystallographic Coxeter groups H2 and H4. These results will enable new applications of group theory in physics (quasicrystals), biology (viruses) and chemistry (fullerenes).

  20. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation. PMID:27367467

  1. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation.

  2. Two measured completely different electron affinities for atomic Eu?

    NASA Astrophysics Data System (ADS)

    Msezane, A. Z.; Felfli, Z.

    2016-05-01

    Recently, the electron affinity (EA) of atomic Eu was measured to be 0.116?eV. This value is in outstanding agreement with the theoretically calculated values using the Regge pole and MCDF-RCI methods. Previously, the EA of Eu was measured to be 1.053 eV. In an attempt to resolve the discrepancy between the two measured values, we have adopted the complex angular momentum (CAM) method and investigated in the electron energy range 0.11 eV value of 2.63 eV as the EA of Eu. This leads us to conclude that neither the claimed measured EA of Eu correspond to the actual EA of Eu. We conclude that the EA in corresponds to the BE of an excited (metastable) state of the Euanion and that in to a shape resonance. We have also investigated the EA of atomic Nd and found the value of 1.88 eV, consistent with the measurement. These significant EA values of Eu and Nd could be important in the use of their negative ions in catalyzing the oxidation of water to peroxide and of methane to methanol without CO2 emission. These new results call for immediate experimental and theoretical verification.

  3. Hot spot detection for breast cancer in Ki-67 stained slides: image dependent filtering approach

    NASA Astrophysics Data System (ADS)

    Niazi, M. Khalid Khan; Downs-Kelly, Erinn; Gurcan, Metin N.

    2014-03-01

    We present a new method to detect hot spots from breast cancer slides stained for Ki67 expression. It is common practice to use centroid of a nucleus as a surrogate representation of a cell. This often requires the detection of individual nuclei. Once all the nuclei are detected, the hot spots are detected by clustering the centroids. For large size images, nuclei detection is computationally demanding. Instead of detecting the individual nuclei and treating hot spot detection as a clustering problem, we considered hot spot detection as an image filtering problem where positively stained pixels are used to detect hot spots in breast cancer images. The method first segments the Ki-67 positive pixels using the visually meaningful segmentation (VMS) method that we developed earlier. Then, it automatically generates an image dependent filter to generate a density map from the segmented image. The smoothness of the density image simplifies the detection of local maxima. The number of local maxima directly corresponds to the number of hot spots in the breast cancer image. The method was tested on 23 different regions of interest images extracted from 10 different breast cancer slides stained with Ki67. To determine the intra-reader variability, each image was annotated twice for hot spots by a boardcertified pathologist with a two-week interval in between her two readings. A computer-generated hot spot region was considered a true-positive if it agrees with either one of the two annotation sets provided by the pathologist. While the intra-reader variability was 57%, our proposed method can correctly detect hot spots with 81% precision.

  4. Therapeutic processes and perceived helpfulness of dang-ki (Chinese shamanism) from the symbolic healing perspective.

    PubMed

    Lee, Boon-Ooi; Kirmayer, Laurence J; Groleau, Danielle

    2010-03-01

    This study focuses on the therapeutic process and perceived helpfulness of dang-ki, a form of Chinese shamanistic healing, in Singapore. It aims to understand the healing symbols employed in dang-ki, whether or not patients find them helpful and whether their perceived helpfulness can be explained by the symbolic healing model (Dow, Am Anthropol 88(1):56-69, 1986; Levi-Strauss, Structural anthropology. Basic Books, New York, 1963). Although many researchers have applied this model to explain the efficacy of shamanistic healings, they did not directly provide empirical support. Furthermore, the therapeutic process of a shared clinical reality as proposed by the model may be achievable in small-scale traditional societies that are culturally more homogeneous than in contemporary societies that are culturally more diversified due to globalization and immigration. Patients may hold multidimensional health belief systems, as biomedicine and alternative healing systems coexist. Thus, it would be interesting to see the relevance and applicability of the symbolic healing model to shamanistic healing in contemporary societies. In this study, ethnographic interviews were conducted with 21 patients over three stages: immediately before and after the healing and approximately 1 month later. The dang-ki healing symbols were identified by observing the healing sessions with video recording. Results show that dang-kis normally applied more than one method to treat a given problem. These methods included words, talismans and physical manipulations. Overall, 11 patients perceived their consultations as helpful, 4 perceived their consultations as helpful but were unable to follow all recommendations, 5 were not sure of the outcome because they had yet to see any concrete results and only 1 patient considered his consultation unhelpful. Although the symbolic healing model provides a useful framework to understand perceived helpfulness, processes such as enactment of a common meaning

  5. Diagnostic implications of Ki-67 expression in adipocytes and lipoblasts: an immunohistochemical study

    PubMed Central

    Wang, Hong-Jun; Li, Cong-Yang; Wang, Yang-Kun

    2014-01-01

    Ki-67 expression is an important tool for distinguishing between malignant and benign tumors. It usually shows the nuclear staining. However, the cell membrane staining of MIB-1, which is one of the clones of Ki-67, in the hyalinizing trabecular adenoma of the thyroid gland and other tumors had also been reported. In our practice, we found that the 7B11 antibody could be immunoreactived with the adipose tissues inside or around tumors in the membrane. Thus, in this study, we determined if Ki-67 expression would be useful in recognizing the lipoblasts and adipocytes. The five clones of the Ki-67 antibody, namely, 7B11, K-2, SP5, MIB-1, and SP6 were selected. The adipocytes showed strong 7B11 staining in the cell membrane. The brown fat cells were strongly immunoreactive with 7B11 in the arachnoid layer of the cytoplasm. The adipocytes and brown fat cells showed positive, albeit weaker K-2 staining in the cell membrane and cytoplasm, respectively, compared to 7B11. The adipose tissues and brown fat cells were non-reactive to clones SP5, MIB-1, and SP6. All adipocytes in the lipomas, angiolipomas, uterine lipoleiomyomas, and angioleiomyolipomas showed diffusedly positive 7B11 and K-2 staining in the cell membrane, with stronger immunoreactivity to 7B11 compared with K-2. All hibernomas showed diffusedly cytoplasmic arachnoid staining of 7B11, but only focal to K-2. The lipoblasts in adipocytic tumors also showed positive 7B11 and K-2 staining; however, nearly all of the vacuolated lipoblasts showed strong 7B11 staining, only focal vacuolated lipoblasts in the adipocytic tumors were immunoreactive to K-2 positivity. All other components of the adipocytic tumors were non-reactive to 7B11, K-2, SP5, MIB-1, and SP6 in the cell membrane and cytoplasm. Our results showed that the 7B11 could well help to identify the lipoblasts, which would be useful to diagnose the malignant adipocytic tumors. PMID:25674262

  6. Deep magnetic anomaly sources interpreted as Otanmäki type Iron ore reserves

    NASA Astrophysics Data System (ADS)

    Korhonen, Juha; Kukkonen, Ilmo

    2013-04-01

    In Otanmäki ore province of Central Finland vertically integrated magnetization is estimated from two aeromagnetic coverages of different altitudes and by varying overall models of regional field. Petrophysically and geochemically determined magnetization of the mined deposits and correlation between it and ore concentration is used to evaluate iron ore reserves in the deeper part of known ore fields. Further, similar analysis is made to nearby magnetically anomalous areas covered by weakly magnetic metasediments, to estimate potential ore reserves at unexposed formations.

  7. Strong Lens Search in the ESO Public Survey KiDS

    NASA Astrophysics Data System (ADS)

    Napolitano, N. R.; Covone, G.; Roy, N.; Tortora, C.; La Barbera, F.; Radovich, M.; Getman, F.; Capaccioli, M.; Colonna, A.; Paolillo, M.; Verdoes Kleijn, G. A.; Koopmans, L. V. E.

    We have started a systematic search for strong lens candidates in the ESO public survey KiDS based on the visual inspection of massive galaxies in the redshift range 0. 1 < z < 0. 5. As a pilot program we have inspected 100 deg2, which overlap with SDSS and where there are known lenses to use as a control sample. Taking advantage of the superb image quality of VST/OmegaCAM, the colour information and accurate model subtracted images, we have found 18 new lens candidates, for which spectroscopic confirmation will be needed to confirm their lensing nature and study the mass profile of the lensing galaxies.

  8. Early Type Galaxies and Structural Parameters from ESO Public Survey KiDS

    NASA Astrophysics Data System (ADS)

    Roy, N.; Napolitano, N. R.; La Barbera, F.; Tortora, C.; Getman, F.; Radovich, M.; Capaccioli, M.

    The Kilo Degree survey (KiDS) is a large-scale optical imaging survey carried out with the VLT Survey Telescope (VST), which is the ideal tool for galaxy evolution studies. We expect to observe millions of galaxies for which we extract the structural parameters in four wavebands (u, g, r and i). This sample will represent the largest dataset with measured structural parameters up to a redshift z = 0. 5. In this paper we will introduce the sample, and describe the 2D fitting procedure using the 2DPHOT environment and the validation of the parameters with an external catalog.

  9. Nordimaprit, homodimaprit, clobenpropit and imetit: affinities for H3 binding sites and potencies in a functional H3 receptor model.

    PubMed

    Kathmann, M; Schlicker, E; Detzner, M; Timmerman, H

    1993-11-01

    We determined the affinities of nordimaprit, homodimaprit, clobenpropit and imetit for H3 binding sites (labelled by 3H-N alpha-methylhistamine) in rat brain cortex homogenates and their potencies at presynaptic H3A receptors on noradrenergic nerve endings in mouse brain cortex slices. 3H-N alpha-Methylhistamine bound saturably to rat brain cortex homogenates with a Kd of 0.70 nmol/l and a Bmax of 98 fmol/mg protein. Binding of 3H-N alpha-methylhistamine was displaced monophasically by dimaprit (pKi 6.55), nordimaprit (5.94), homodimaprit (6.44), clobenpropit (9.16), imetit (9.83), R-(-)-alpha-methylhistamine (8.87) and histamine (8.20), and biphasically by burimamide (pKi high 7.73, pKi low 5.97). In superfused mouse brain cortex slices preincubated with 3H-noradrenaline, the electrically (0.3 Hz) evoked tritium overflow was inhibited by imetit (pIC35 8.93), R-(-)-alpha-methylhistamine (7.87) and histamine (7.03). The effect of histamine was attenuated by nordimaprit, homodimaprit, clobenpropit and N-ethoxycarbonyl-2- ethoxy-1,2-dihydroquinoline (EEDQ); EEDQ (but not nordimaprit, homodimaprit and clobenpropit) attenuated the effect of histamine also in slices pre-exposed to the drug 60-30 min prior to superfusion. The concentration-response curve of histamine was shifted to the right by homodimaprit and clobenpropit; Schild plots yielded straight lines with a slope of unity for both drugs (pA2 5.94 and 9.55, respectively). Nordimaprit depressed the maximum effect of histamine (pD'2 5.55) and also slightly increased the concentration of histamine producing the half-maximum effect.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Smooth big bounce from affine quantization

    NASA Astrophysics Data System (ADS)

    Bergeron, Hervé; Dapor, Andrea; Gazeau, Jean Pierre; Małkiewicz, Przemysław

    2014-04-01

    We examine the possibility of dealing with gravitational singularities on a quantum level through the use of coherent state or wavelet quantization instead of canonical quantization. We consider the Robertson-Walker metric coupled to a perfect fluid. It is the simplest model of a gravitational collapse, and the results obtained here may serve as a useful starting point for more complex investigations in the future. We follow a quantization procedure based on affine coherent states or wavelets built from the unitary irreducible representation of the affine group of the real line with positive dilation. The main issue of our approach is the appearance of a quantum centrifugal potential allowing for regularization of the singularity, essential self-adjointness of the Hamiltonian, and unambiguous quantum dynamical evolution.

  11. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  12. Artificial Affinity Proteins as Ligands of Immunoglobulins

    PubMed Central

    Mouratou, Barbara; Béhar, Ghislaine; Pecorari, Frédéric

    2015-01-01

    A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized. PMID:25647098

  13. Assessment of early changes in 3H-fluorothymidine uptake after treatment with gefitinib in human tumor xenograft in comparison with Ki-67 and phospho-EGFR expression

    PubMed Central

    2013-01-01

    Background The purpose of this study was to evaluate whether early changes in 3′-deoxy-3′-3H-fluorothymidine (3H-FLT) uptake can reflect the antiproliferative effect of gefitinib in a human tumor xenograft, in comparison with the histopathological markers, Ki-67 and phosphorylated EGFR (phospho-EGFR). Methods An EGFR-dependent human tumor xenograft model (A431) was established in female BALB/c athymic mice, which were divided into three groups: one control group and two treatment groups. Mice in the treatment groups were orally administered a partial regression dose (100 mg/kg/day) or the maximum tolerated dose of gefitinib (200 mg/kg/day), once daily for 2 days. Mice in the control group were administered the vehicle (0.1% Tween 80). Tumor size was measured before and 3 days after the start of treatment. Biodistribution of 3H-FLT and 18F-FDG (%ID/g/kg) was examined 3 days after the start of the treatment. Tumor cell proliferative activity with Ki-67 was determined. Immunohistochemical staining of EGFR and measurement of phospho-EGFR were also performed. Results High expression levels of EGFR and Ki-67 were observed in the A431 tumor. After the treatment with 100 and 200 mg/kg gefitinib, the uptake levels of 3H-FLT in the tumor were significantly reduced to 67% and 61% of the control value, respectively (0.39 ± 0.09, 0.36 ± 0.06, 0.59 ± 0.11%ID/g/kg for 100 mg/kg, 200 mg/kg, and control groups, respectively; p < 0.01 vs. control), but those of 18F-FDG were not. After the treatment with 100 and 200 mg/kg gefitinib, the expression levels of Ki-67 in the tumor were markedly decreased (4.6 ± 2.4%, 6.2 ± 1.8%, and 10.4 ± 5.7% for 100 mg/kg, 200 mg/kg, and control groups, respectively, p < 0.01 vs. control). The expression levels of the phospho-EGFR protein also significantly decreased (29% and 21% of the control value for 100, and 200 mg/kg, respectively p < 0.01 vs. control). There was no statistically

  14. Permeability of self-affine rough fractures

    PubMed

    Drazer; Koplik

    2000-12-01

    The permeability of two-dimensional fractures with self-affine fractal roughness is studied via analytic arguments and numerical simulations. The limit where the roughness amplitude is small compared with average fracture aperture is analyzed by a perturbation method, while in the opposite case of narrow aperture, we use heuristic arguments based on lubrication theory. Numerical simulations, using the lattice Boltzmann method, are used to examine the complete range of aperture sizes, and confirm the analytic arguments. PMID:11138092

  15. Theoretical study for the electron affinities of negative ions with the MCDHF method

    NASA Astrophysics Data System (ADS)

    Li, Junqin; Zhao, Zilong; Andersson, Martin; Zhang, Xuemei; Chen, Chongyang

    2012-08-01

    Systematic theoretical calculations based on the multi-configuration Dirac-Hartree-Fock method have been carried out for the electron affinities of anions of the elements of group III (B, Al, Ga, In and Tl), group IV (C, Si, Ge, Sn and Pb), group V (N, P and As), group VI (O, S, Se, Te and Po) and group VII (F, Cl, Br, I and At) by studying the ground energies of neutral atoms and their corresponding negative ions. The differences between the calculated total energies of the neutral atom and its anion were used to obtain the electron affinities. We discuss in detail the effects of configuration interaction, investigate the importance of including different types of correlations and check the impact of the higher order relativistic corrections on electron affinities. Our calculated electron affinities are compared with experimental and other available theoretical results. The present studies are the first systematic studies of all these elements. We give the first theoretical values for the affinities of elements Se, Te, Po and At; thereinto, there is no experimental value for elements Po and At.

  16. Flow cytometric analysis of DNA content and Ki-67-positive fractions in the diagnosis of salivary gland tumors.

    PubMed

    Horii, A; Yoshida, J; Sakai, M; Okamoto, S; Kubo, T

    1998-01-01

    To explore the utility of flow cytometry (FCM) for the diagnosis of histopathology of salivary gland tumors, fresh materials taken at surgery from 23 Warthin's tumors, 57 pleomorphic adenomas, and 14 malignant tumors were analyzed for DNA ploidy and proliferative cell activities, including S-phase fraction (SPF), G2- plus M-phase fraction (G2M), and Ki-67-positive fraction. To facilitate this study, glands were taken from all major salivary sites and minor glands in the head and neck. DNA aneuploidy was not detected in the benign tumors. Nine of 14 malignant tumors showed DNA aneuploidy. The percentage of SPF or G2M of the malignant tumors was significantly higher than those of the benign tumors. The percentage of Ki-67-positive fraction of pleomorphic adenomas was comparable to that of malignant tumors and was significantly higher than that of Warthin's tumors. Ki-67 of 20% as a cut-off had a sensitivity of 88%, specificity of 100%, and accuracy of 91% for differentiating pleomorphic adenomas from Warthin's tumors. In analyzing DNA content and proliferative activities by FCM, we could distinguish among the three major histopathologies of salivary gland tumors. Warthin's tumors showed low SPF + G2M with low Ki-67, pleomorphic adenomas had low SPF + G2M with high Ki-67, and malignant tumor showed high SPF + G2M with high Ki-67. The high percentage of the Ki-67-positive fraction seen in pleomorphic adenomas may reflect their potential biological aggressiveness manifested as tumor recurrence or malignant transformation.

  17. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  18. Overview of affinity biosensors in food analysis.

    PubMed

    Patel, Pradip D

    2006-01-01

    The 4 major driving forces that are expected to lead to increased use of affinity biosensors that meet crucial industrial test specifications, e.g., fast, reliable, cost-effective, and use of low-skilled personnel, are (1) strict legislative framework, e.g., recent changes proposed to the European food safety and hygiene legislation, EC No. 178/2002; (2) industrial shift from quality control to quality assurance procedures, e.g., Hazard Analysis Critical Control Point, ensuring effective positioning in the global competitive trade; (3) just-in-time production resulting in 'right' product every time; and (4) consumer demand for safe and wholesome products. The affinity biosensors field has expanded significantly over the past decade, with a projected global biosensors market growth from $6.1 billion in 2004 to $8.2 billion in 2009, representing major industrial sectors (e.g., Pharma, Medicare, and Food). This brief review is targeted to affinity biosensors developed for the food industry and includes research and development leading to biosensors for microbiological and chemical analytes of industrial concern, commercial biosensors products on the market, and examples of future prospects in this diagnostic field.

  19. Overview of affinity biosensors in food analysis.

    PubMed

    Patel, Pradip D

    2006-01-01

    The 4 major driving forces that are expected to lead to increased use of affinity biosensors that meet crucial industrial test specifications, e.g., fast, reliable, cost-effective, and use of low-skilled personnel, are (1) strict legislative framework, e.g., recent changes proposed to the European food safety and hygiene legislation, EC No. 178/2002; (2) industrial shift from quality control to quality assurance procedures, e.g., Hazard Analysis Critical Control Point, ensuring effective positioning in the global competitive trade; (3) just-in-time production resulting in 'right' product every time; and (4) consumer demand for safe and wholesome products. The affinity biosensors field has expanded significantly over the past decade, with a projected global biosensors market growth from $6.1 billion in 2004 to $8.2 billion in 2009, representing major industrial sectors (e.g., Pharma, Medicare, and Food). This brief review is targeted to affinity biosensors developed for the food industry and includes research and development leading to biosensors for microbiological and chemical analytes of industrial concern, commercial biosensors products on the market, and examples of future prospects in this diagnostic field. PMID:16792079

  20. A MEMS Dielectric Affinity Glucose Biosensor

    PubMed Central

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-01-01

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concentrations. This sensor features simplicity in sensor design, and possesses high specificity and accuracy in glucose detection. However, lack of glucose diffusion passage, this device is unable to fulfill real-time in-vivo monitoring. As a major improvement to this device, we present in this paper a fully implantable MEMS dielectric affinity glucose biosensor that contains a perforated electrode embedded in a suspended diaphragm. This capacitive-based sensor contains no moving parts, and enables glucose diffusion and real-time monitoring. The experimental results indicate that this sensor can detect glucose solutions at physiological concentrations and possesses good reversibility and reliability. This sensor has a time constant to glucose concentration change at approximately 3 min, which is comparable to commercial systems. The sensor has potential applications in fully implantable CGM that require excellent long-term stability and reliability. PMID:24511215

  1. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  2. Trematode hemoglobins show exceptionally high oxygen affinity.

    PubMed

    Kiger, L; Rashid, A K; Griffon, N; Haque, M; Moens, L; Gibson, Q H; Poyart, C; Marden, M C

    1998-08-01

    Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.

  3. Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

    PubMed

    Ball, D J; Nishimura, J S

    1980-11-25

    Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits. PMID:7430155

  4. Ki67 index is an independent prognostic factor in epithelioid but not in non-epithelioid malignant pleural mesothelioma: a multicenter study

    PubMed Central

    Ghanim, B; Klikovits, T; Hoda, M A; Lang, G; Szirtes, I; Setinek, U; Rozsas, A; Renyi-Vamos, F; Laszlo, V; Grusch, M; Filipits, M; Scheed, A; Jakopovic, M; Samarzija, M; Brcic, L; Stancic–Rokotov, D; Kern, I; Rozman, A; Dekan, G; Klepetko, W; Berger, W; Glasz, T; Dome, B; Hegedus, B

    2015-01-01

    Background: Estimating the prognosis in malignant pleural mesothelioma (MPM) remains challenging. Thus, the prognostic relevance of Ki67 was studied in MPM. Methods: Ki67 index was determined in a test cohort of 187 cases from three centres. The percentage of Ki67-positive tumour cells was correlated with clinical variables and overall survival (OS). The prognostic power of Ki67 index was compared with other prognostic factors and re-evaluated in an independent cohort (n=98). Results: Patients with Ki67 higher than median (>15%) had significantly (P<0.001) shorter median OS (7.5 months) than those with low Ki67 (19.1 months). After multivariate survival analyses, Ki67 proved to be—beside histology and treatment—an independent prognostic marker in MPM (hazard ratio (HR): 2.1, P<0.001). Interestingly, Ki67 was prognostic exclusively in epithelioid (P<0.001) but not in non-epithelioid subtype. Furthermore, Ki67 index was significantly lower in post-chemotherapy samples when compared with chemo-naive cases. The prognostic power was comparable to other recently published prognostic factors (CRP, fibrinogen, neutrophil-to-leukocyte ratio (NLR) and nuclear grading score) and was recapitulated in the validation cohort (P=0.048). Conclusion: This multicentre study demonstrates that Ki67 is an independent and reproducible prognostic factor in epithelioid but not in non-epithelioid MPM and suggests that induction chemotherapy decreases the proliferative capacity of MPM. PMID:25633038

  5. Associations of Teacher Credibility and Teacher Affinity with Learning Outcomes in Health Classrooms

    ERIC Educational Resources Information Center

    Gray, DeLeon L.; Anderman, Eric M.; O'Connell, Ann A.

    2011-01-01

    In the present study (N = 633), we examine the role of teacher credibility and teacher affinity in classrooms. We explore the relations among these two characteristics and student gains in knowledge and valuing of learning about HIV and pregnancy prevention across high school classrooms. Results marshaled support for the notion that teacher…

  6. Basic FGF and Ki-67 proteins useful for immunohistological diagnostic evaluations in malignant solitary fibrous tumor.

    PubMed

    Sun, Yuliang; Naito, Zenya; Ishiwata, Toshiyuki; Maeda, Shotaro; Sugisaki, Yuichi; Asano, Goro

    2003-05-01

    Solitary fibrous tumor (SFT) is an uncommon soft tissue tumor initially reported in the pleura but recently described in other sites in the body. Morphological distinction between benign and malignant SFT is often difficult. An immunohistochemical study was performed in pleural and extrapleural sites. The aim of this study was to determine if an immunohistochemical method is helpful in distinguishing benign SFT from malignant SFT, and providing valid information to predict the prognosis associated with malignant SFT. Twenty-four cases of benign (14 patients) and malignant (10 patients) SFT in the pleura, pelvic space, prostate and other sites of soft tissue were analyzed. Tumors from 10 patients were diagnosed as malignant on the basis of markedly increased cellularity, mitotic activity (>4/10 high-power fields), nuclear pleomorphism and areas of necrosis. Immunohistochemically, we found a mean basic fibroblast growth factor (bFGF) labeling index of 48.67% (48.67 +/- 8.52%) for benign SFT and 74.5% (74.5 +/- 6.92%) for malignant SFT (P < 0.05). We also found a mean Ki-67 labeling index of 1.9% (1.9 +/- 0.43%) for benign SFT and 6.11% (6.11 +/- 1.05%) for malignant SFT (P < 0.05). Our results suggest that bFGF and Ki-67 are diagnostically relevant to the evaluation of malignant SFT and these proteins are thought to be potentially useful markers for prognosis of SFT.

  7. A re-examination of the human fossil specimen from Bački Petrovac (Serbia).

    PubMed

    Radović, Predrag; Lindal, Joshua Allan; Roksandic, Mirjana

    2014-08-01

    A fragmented human calotte was discovered during the early 1950s near Bački Petrovac (Serbia), in association with Palaeolithic stone tools. After its initial publication, the fossil specimen remained largely unknown outside of the Serbian academe and no detailed comparative study has ever been carried out. Since the whereabouts of the fossil itself are currently unknown, and given its potential significance for the Pleistocene human evolution, we re-examine the data published by Živanović (1966, 1975). Using the original measurements, mostly taken on the frontal bone, and a wide comparative sample of 68 fossil specimens, the fossil was compared and analyzed by statistical multivariate methods. We also conducted a visual examination of the morphology based on the available photographic material. Our analysis reveals phenetic similarity with Middle Pleistocene archaic Homo from Africa and anatomically modern Homo sapiens. However, the absence of primitive cranial traits in Bački Petrovac indicates a clear modern Homo sapiens designation. Although lost at the moment, there is a chance for the re-discovery of the fossil in the years to come. This would give us an opportunity to acquire absolute dates and to study the specimen in a more detailed manner.

  8. Nonstoichiometric complex of gramicidin D with KI at 0.80 [angstrom] resolution

    SciTech Connect

    Olczak, A.; Glowka, M.L.; Szczesio, M.; Bojarsk, J.; Duax, W.L.; Burkhart, B.M.; Wawrzak, Z.

    2010-03-08

    The crystal structure of a nonstoichiometric complex of gramicidin D (gD) with KI has been determined at 100 K using synchrotron radiation. The final R factor was 0.106 for 83 988 observed reflections (Friedel pairs were not merged) collected to 0.80 {angstrom}. The structure consists of four independent pentadecapeptides and numerous solvent molecules and salt ions. The general architecture of the antiparallel double-stranded gramicidin dimers in the crystal (a right-handed antiparallel DS{beta}H{sub R} form) closely resembles that of previously published cation complexes of gD. However, a significantly different mixture of gramicidin isomers is found in the crystal of the KI complex, including partial occupancy of phenylalanine at position 11. Only three sites in each of the two crystallographically independent channels are partially occupied by potassium cations instead of the commonly observed seven sites. The sum of the partial occupancies of K{sup +} (1.10 per two dimers) is consistent with the sum of the iodide occupancies (1.095 over eight sites), which is also confirmed by the anomalous signal of the iodide. There was a significant asymmetry of the distribution and occupancies of cations in the crystallographically independent gramicidin channels, in contrast to the distribution found in the rubidium chloride complex with gD.

  9. Sialoblastoma: utility of Ki-67 and p53 as a prognostic tool and review of literature.

    PubMed

    Patil, Deepa T; Chou, Pauline M

    2010-01-01

    Sialoblastoma is a rare tumor of the salivary gland that commonly occurs in the parotid gland and occasionally in the sub-mandibular gland. The malignant potential of sialoblastoma has been documented in only 3 of 32 cases of sialoblastoma reported thus far. In the last 15 years, we have encountered 2 cases of sialoblastoma, in a newborn and in a 15-year-old boy, both arising within the parotid gland. Case 1 has been previously reported and although there were 2 recurrences, at 1 and 9 years post resection, it has shown benign biological behavior. Case 2 is unusual since the patient presented with metastases. We reviewed the 2 cases, including the 2 recurrences from the first case, for histologic and immunohistochemical differences. Although both cases showed similar cytomorphologic features, there was a significant difference in Ki-67 expression: 20% in case 1 (original tumor), <2% in case 1 (recurrent tumor), and nearly 70-80% in the recent malignant case. The difference is remarkable when combined with p53 expression, which was focally positive in the first case but diffusely positive in the second. This report highlights the potential utility of proliferation markers such as Ki-67 in concert with p53 expression to better predict the biological behavior of a rare but locally aggressive neoplasm.

  10. Effects of whole-body irradiation on antibody affinity.

    PubMed Central

    Gorini, G; Adorini, L; Boraschi, D; Di Michele, A; Doria, G

    1977-01-01

    Mice exposed to a sublethal dose of X-rays were immunized with alum-precipitated DNP-KLH (dinitrophenyl-keyhole limpet haemocyanin) and B. pertussis either before or after irradiation. The primary anti-DNP antibody response was evaluated during 8 weeks after immunization by the equilibrium dialysis technique using ammonium sulphate- precipitated serum globulins and the ligand 3H-labelled xi-DNP-L-Lysine. The serum concentrations of antibody sites in mice immunized 1-5 days before or 2 h-8 weeks after 450 rad were below the values in unirradiated controls at all bleeding times. Antibody affinity, however, was found to be up to 20 fold higher in irradiated mice than in control mice when antigen was injected before, or 3-8 weeks after, irradiation. Spleen cells from mice exposed to 450 rad 1-9 weeks before killing were stimulated in vitro with PHA, ConA, or LPS. Recovery profiles of mitotic responsiveness suggest that enhancement of antibody affinity in irradiated mice could result from relative lack of suppressor T Cells. PMID:198358

  11. Boronate affinity adsorption of RNA: possible role of conformational changes

    NASA Technical Reports Server (NTRS)

    Singh, N.; Willson, R. C.; Fox, G. E. (Principal Investigator)

    1999-01-01

    Batch equilibrium adsorption isotherm determination is used to characterize the adsorption of mixed yeast RNA on agarose-immobilized m-aminophenylboronic acid. It is shown that the affinity-enhancing influence of divalent cations depends strongly on the precise nature of the cation used, with barium being far more effective than the conventionally-used magnesium. This adsorption-promoting influence of barium is suggested to arise primarily from ionic influences on the structure and rigidity of the RNA molecule, as the adsorption of ribose-based small molecules is not similarly affected. The substitution of barium for the standard magnesium counterion does not greatly promote the adsorption of DNA, implying that the effect is specific to RNA and may be useful in boronate-based RNA separations. RNA adsorption isotherms exhibit sharp transitions as functions of temperature, and these transitions occur at different temperatures with Mg2+ and with Ba2+. Adsorption affinity and capacity were found to increase markedly at lower temperatures, suggestive of an enthalpically favored interaction process. The stoichiometric displacement parameter, Z, in Ba2+ buffer is three times the value in Mg2+ buffer, and is close to unity.

  12. Characterization of methacrylate chromatographic monoliths bearing affinity ligands.

    PubMed

    Černigoj, Urh; Vidic, Urška; Nemec, Blaž; Gašperšič, Jernej; Vidič, Jana; Lendero Krajnc, Nika; Štrancar, Aleš; Podgornik, Aleš

    2016-09-16

    We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and various affinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant nonlinear effect of ligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12mg/mL exceeding theoretical monolayer adsorption value of 7.8mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacity linearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher. PMID:27554023

  13. Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity.

    PubMed

    M'Barek, Sarrah; Chagot, Benjamin; Andreotti, Nicolas; Visan, Violeta; Mansuelle, Pascal; Grissmer, Stephan; Marrakchi, Mohamed; El Ayeb, Mohamed; Sampieri, François; Darbon, Hervé; Fajloun, Ziad; De Waard, Michel; Sabatier, Jean-Marc

    2005-08-15

    Scorpion toxins interact with their target ion channels through multiple molecular contacts. Because a "gain of function" approach has never been described to evaluate the importance of the molecular contacts in defining toxin affinity, we experimentally examined whether increasing the molecular contacts between a toxin and an ion channel directly impacts toxin affinity. For this purpose, we focused on two scorpion peptides, the well-characterized maurotoxin with its variant Pi1-like disulfide bridging (MTX(Pi1)), used as a molecular template, and butantoxin (BuTX), used as an N-terminal domain provider. BuTX is found to be 60-fold less potent than MTX(Pi1) in blocking Kv1.2 (IC(50) values of 165 nM for BuTX versus 2.8 nM for MTX(Pi1)). Removal of its N-terminal domain (nine residues) further decreases BuTX affinity for Kv1.2 by 5.6-fold, which is in agreement with docking simulation data showing the importance of this domain in BuTX-Kv1.2 interaction. Transfer of the BuTX N-terminal domain to MTX(Pi1) results in a chimera with five disulfide bridges (BuTX-MTX(Pi1)) that exhibits 22-fold greater affinity for Kv1.2 than MTX(Pi1) itself, in spite of the lower affinity of BuTX as compared to MTX(Pi1). Docking experiments performed with the 3-D structure of BuTX-MTX(Pi1) in solution, as solved by (1)H-NMR, reveal that the N-terminal domain of BuTX participates in the increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicate that acting on molecular contacts between a toxin and a channel is an efficient strategy to modulate toxin affinity.

  14. Calculation of the Ionization Potentials and Electron Affinities for Atoms

    NASA Astrophysics Data System (ADS)

    Chen, Jiqiang; Krieger, J. B.; Iafrate, G. J.; Savin, A.

    1998-03-01

    The method employing the self-interaction-corrected correlation energy functional obtained from the homogeneous electron gas with a gap is extended to atoms and ions with non-zero spin polarization. As in the case for atoms and ions with ζ=3D0, the error in the calculated Ec is significantly smaller than in the LSD approximation with zero gap for atoms and ions with Z<=18. Comparison of the resulting ionization potentials and electron affinities with experimental values will also be presented. Finally, we will discuss the possibility of obtaining saturation for Ec for the He, Li, N, O, F and Ne isoelectronic series, but a divergent Ec for the Be, B and C isoelectronic series, in the large Z limit.

  15. Class II-restricted T cell receptor engineered in vitro for higher affinity retains peptide specificity and function

    PubMed Central

    Weber, K. Scott; Donermeyer, David L.; Allen, Paul M.; Kranz, David M.

    2005-01-01

    The T cell receptor (TCR) αβ heterodimer determines the peptide and MHC specificity of a T cell. It has been proposed that in vivo selection processes maintain low TCR affinities because T cells with higher-affinity TCRs would (i) have reduced functional capacity or (ii) cross-react with self-peptides resulting in clonal deletion. We used the class II-restricted T cell clone 3.L2, specific for murine hemoglobin (Hb/I-Ek), to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR (Vβ-linker-Vα) was mutagenized and selected for thermal stability and surface expression in a yeast display system. Stabilized mutants were used to generate a library with CDR3 mutations that were selected with Hb/I-Ek to isolate a panel of affinity mutants with KD values as low as 25 nM. Kinetic analysis of soluble single-chain TCRs showed that increased affinities were the result of both faster on-rates and slower off-rates. T cells transfected with the mutant TCRs and wild-type TCR responded to similar concentrations of peptide, indicating that the increased affinity was not detrimental to T cell activation. T cell transfectants maintained exquisite hemoglobin peptide specificity, but an altered peptide ligand that acted as an antagonist for the wild-type TCR was converted to a strong agonist with higher-affinity TCRs. These results show that T cells with high-affinity class II reactive TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of T cell activation. PMID:16365315

  16. Latest European coelacanth shows Gondwanan affinities.

    PubMed

    Cavin, Lionel; Forey, Peter L; Buffetaut, Eric; Tong, Haiyan

    2005-06-22

    The last European fossil occurrence of a coelacanth is from the Mid-Cretaceous of the English Chalk (Turonian, 90 million years ago). Here, we report the discovery of a coelacanth from Late Cretaceous non-marine rocks in southern France. It consists of a left angular bone showing structures that imply close phylogenetic affinities with some extinct Mawsoniidae. The closest relatives are otherwise known from Cretaceous continental deposits of southern continents and suggest that the dispersal of freshwater organisms from Africa to Europe occurred in the Late Cretaceous.

  17. On the structure of self-affine convex bodies

    SciTech Connect

    Voynov, A S

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  18. Measuring an antibody affinity distribution molecule by molecule.

    PubMed

    Temirov, Jamshid P; Bradbury, Andrew R M; Werner, James H

    2008-11-15

    Single molecule fluorescence microscopy was used to observe the binding and unbinding of hapten decorated quantum dots to individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  19. Measuring an antibody affinity distribution molecule by molecule

    SciTech Connect

    Bradbury, Andrew M; Werner, James H; Temirov, Jamshid

    2008-01-01

    Single molecule fluorescence mIcroscopy was used to observe the binding and unbinding of hapten decorated quantum dots with individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  20. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

  1. Current Status and Future Prospect of K-NET and KiK-net

    NASA Astrophysics Data System (ADS)

    Aoi, S.; Kunugi, T.; Suzuki, W.; Nakamura, H.; Fujiwara, H.

    2014-12-01

    During 18 years since the deployment of K-NET following the Kobe earthquake, our attention has mainly focused on rapidity of the data collection and an unfailing and reliable observation. In this presentation, we review three generations of the instruments employed by K-NET and KiK-net from these two points of view.At beginning of the 2000's, we newly developed the second generation instruments (K-NET02, K-NET02A, KiK-net06) to replace the first generation instruments (K-NET95, SMAC-MDK) employed when the networks were constructed in the 1990's. These instruments have an automatic dial-out function. It takes typically 2-5 s to establish communication and a few seconds to send the pre-trigger data. After that, data is available typically within a 1.5 s delay. Not only waveform data but also strong motion indexes such as real-time intensity, PGA, PGV, PGD, and response spectra are continuously sent once a second.After the 2011 Tohoku earthquake, we have developed the third generation instruments (K-NET11, KiK-net11) and have replaced almost half of the all stations country wide. Main improvement of this instrument is more unfailing and reliable observation. Because we have often experienced very large ground motions (e.g. 45 records exceeding gravity), the maximum measureable range was expanded from 2000 gal to 4000 gal for the second generation instrument, and to 8000 gal for the third. For the third generation instrument, in case of power failure, observation (including transmission of data) works for seven days thanks to the backup battery, while for the second generation instruments it works only for one day. By adding an oblique component to the three-component accelerometers, we could automatically distinguish shaking data from noise such as electric pulses which may cause a false alarm in EEW. Implementation to guarantee the continuity of observation under severe conditions such as during the Tohoku earthquake is very important, as well as a highly efficient

  2. [Affine transformation-based automatic registration for peripheral digital subtraction angiography (DSA)].

    PubMed

    Kong, Gang; Dai, Dao-Qing; Zou, Lu-Min

    2008-07-01

    In order to remove the artifacts of peripheral digital subtraction angiography (DSA), an affine transformation-based automatic image registration algorithm is introduced here. The whole process is described as follows: First, rectangle feature templates are constructed with their centers of the extracted Harris corners in the mask, and motion vectors of the central feature points are estimated using template matching technology with the similarity measure of maximum histogram energy. And then the optimal parameters of the affine transformation are calculated with the matrix singular value decomposition (SVD) method. Finally, bilinear intensity interpolation is taken to the mask according to the specific affine transformation. More than 30 peripheral DSA registrations are performed with the presented algorithm, and as the result, moving artifacts of the images are removed with sub-pixel precision, and the time consumption is less enough to satisfy the clinical requirements. Experimental results show the efficiency and robustness of the algorithm.

  3. Work function and electron affinity of the fluorine-terminated (100) diamond surface

    NASA Astrophysics Data System (ADS)

    Rietwyk, K. J.; Wong, S. L.; Cao, L.; O'Donnell, K. M.; Ley, L.; Wee, A. T. S.; Pakes, C. I.

    2013-03-01

    The work function and electron affinity of fluorine-terminated (100) diamond surfaces prepared by exposure to dissociated XeF2 have been determined using synchrotron-based photoemission. After vacuum annealing to 350 °C a clean, monofluoride terminated C(100):F surface was obtained for which an electron affinity of 2.56 eV was measured. This is the highest electron affinity reported for any diamond surface termination so far, and it exceeds the value predicted by recent density functional theory calculations by 0.43 eV. The work function of 7.24 eV measured for the same surface places the Fermi energy of 0.79 eV above the valence band maximum.

  4. Soybean. beta. -glucan binding sites display maximal affinity for a heptaglucoside phytoalexin-elicitor

    SciTech Connect

    Cosio, E.G.; Waldmueller, T.; Frey, T.; Ebel, J. )

    1990-05-01

    The affinity of soybean {beta}-glucan-binding sites for a synthetic heptaglucan elicitor was tested in a ligand-competition assay against a {sup 125}I-labeled 1,3-1,6-{beta}-glucan preparation (avg. DP=20). Half-maximal displacement of label (IC{sub 50}) was obtained at 9nM heptaglucan, the highest affinity of all fractions tested to date. Displacement followed a uniform sigmoidal pattern and was complete at 1{mu}M indicating access of heptaglucan to all sites available to the labeled elicitor. A mathematical model was used to predict IC{sub 50} values according to the DP of glucan fragments obtained from fungal cell walls. The lowest IC{sub 50} predicted by this model is 3nM. Binding affinity of the glucans was compared with their elicitor activity in a bioassay.

  5. Optimized Structures and Proton Affinities of Fluorinated Dimethyl Ethers: An Ab Initio Study

    NASA Technical Reports Server (NTRS)

    Orgel, Victoria B.; Ball, David W.; Zehe, Michael J.

    1996-01-01

    Ab initio methods have been used to investigate the proton affinity and the geometry changes upon protonation for the molecules (CH3)2O, (CH2F)2O, (CHF2)2O, and (CF3)2O. Geometry optimizations were performed at the MP2/3-2 I G level, and the resulting geometries were used for single-point energy MP2/6-31G calculations. The proton affinity calculated for (CH3)2O was 7 Kjoule/mole from the experimental value, within the desired variance of +/- 8Kjoule/mole for G2 theory, suggesting that the methodology used in this study is adequate for energy difference considerations. For (CF3)20, the calculated proton affinity of 602 Kjoule/mole suggests that perfluorinated ether molecules do not act as Lewis bases under normal circumstances; e.g. degradation of commercial lubricants in tribological applications.

  6. Avoiding degenerate coframes in an affine gauge approach to quantum gravity

    SciTech Connect

    Mielke, E.W.; McCrea, J.D.; Ne`eman, Y.; Hehl, F.W.

    1993-04-01

    This report discusses the following concepts on quantum gravity: The affine gauge approach; affine gauge transformations versus active differomorphisms; affine gauge approach to quantum gravity with topology change.

  7. Proton and sodium cation affinities of harpagide: a computational study.

    PubMed

    Colas, Cyril; Bouchonnet, Stéphane; Rogalewicz-Gilard, Françoise; Popot, Marie-Agnès; Ohanessian, Gilles

    2006-06-15

    The aim of this work was to estimate the proton and sodium cation affinities of harpagide (Har), an iridoid glycoside responsible for the antiinflammatory properties of the medicinal plant Harpagophytum. Monte Carlo conformational searches were performed at the semiempirical AM1 level to determine the most stable conformers for harpagide and its protonated and Na+-cationized forms. The 10 oxygen atoms of the molecule were considered as possible protonation and cationization sites. Geometry optimizations were then refined at the DFT B3LYP/6-31G level from the geometries of the most stable conformers found. Final energetics were obtained at the B3LYP/6-311+G(2d,2p)//B3LYP/6-31G level. The proton and sodium ion affinities of harpagide have been estimated at 223.5 and 66.0 kcal/mol, respectively. Since harpagide mainly provides HarNa+ ions in electrospray experiments, the DeltarG298 associated with the reaction of proton/sodium exchange between Har and methanol, MeOHNa+ + HarH+ --> MeOH2+ + HarNa+ (1), has been calculated; it has been estimated to be 1.9 kcal/mol. Complexing a methanol molecule to each reagent and product of reaction 1 makes the reaction become exothermic by 1.7 kcal/mol. These values are in the limit of the accuracy of the method and do not allow us to conclude definitely whether the reaction is endo- or exothermic, but, according to these very small values, the cation exchange reaction is expected to proceed easily in the final stages of the ion desolvation process. PMID:16759142

  8. Chemical and radiological characterization of fly and bottom ash landfill of the former sulfate pulp factory Plaški and its surroundings.

    PubMed

    Oreščanin, Višnja; Kollar, Robert; Buben, Kresimir; Mikelic, Ivanka Lovrencic; Kollar, Karlo; Kollar, Melkior; Medunic, Gordana

    2012-01-01

    The subject of this study was chemical and radiological characterization of the fly and bottom ash, by-product of the combustion of coal used as an energy source in the former sulfate pulp factory in Plaški. The research involves determination of the concentration of macro, micro and trace elements and activities of the radionuclides in: (i) ash from different positions of the landfill; (ii) soil samples in the zone of the influence of the landfill; (iii) control soil samples and (iv) sediment sample from the river Dretulja. Besides, in situ measurement of an effective dose rate above ash/soil was also determined. In relation with the control soil the average increase of the concentrations of the elements Ca, Cd, Hg, Ni, Se, Sr, Th and U in the samples taken from the fly and bottom ash landfill as well as soil samples within the radius of 300 m from the landfill was 38.3, 6.7, 9.9, 8.5, 9.4, 7.2, 3.6 and 5.7 times, respectively. In these samples, the concentrations of the above mentioned elements were in the following ranges: calcium from 7.94 to 19.7 %; cadmium from 0.33 to 1.66 mg/kg; mercury from 0.18 to 0.49 mg/kg; nickel from 260 to 1500 mg/kg; selenium from 2.7 to 21 mg/kg; strontium from 176 to 542 mg/kg; thorium from 8 to 55 mg/kg and uranium from 5.6 to 19.7 mg/kg. Compared to the world's average soil concentration, uranium and thorium values increased 3.7 and 1.7 times, respectively. The mean value of the total effective dose rate measured in the air at the height of 1 m for all samples of ash and soil under the influence of the landfill was 1.60 mSv/yr. Compared to the Croatian average (0.7015 mSv/yr), the determined mean value for the Plaški landfill is two times higher. However, compared to the local background (0.14 mSv/yr), the mean value of the total effective dose rate measured above the Plaški landfill is 11.4 times higher. In the samples of ash and contaminated soil regardless of the sampling location the activity concentrations of the

  9. Prognostic relevance of Ki-67 in the primary tumor for survival after a diagnosis of distant metastasis.

    PubMed

    Loehberg, Christian R; Almstedt, Katrin; Jud, Sebastian M; Haeberle, Lothar; Fasching, Peter A; Hack, Carolin C; Lux, Michael P; Thiel, Falk C; Schrauder, Michael G; Brunner, Michaela; Bayer, Christian M; Hein, Alexander; Heusinger, Katharina; Heimrich, Jutta; Bani, Mayada R; Renner, Stefan P; Hartmann, Arndt; Beckmann, Matthias W; Wachter, David L

    2013-04-01

    Prediction of the prognosis for metastatic breast cancer patients depends on molecular subtypes similar to those found in patients with primary breast cancer. Several studies have shown that estrogen receptor (ER) and progesterone receptor (PR) status determine the course of the disease and the prognosis. As Ki-67 helps to differentiate molecular subtypes in patients with primary breast cancer, the aim of this study was to assess the prognostic relevance of Ki-67 in the primary tumor in relation to its prognostic relevance for patients with metastatic breast cancer. A total of 467 patients with invasive breast cancer were identified in the database of a single breast cancer center, in whom Ki-67 had been assessed in tumor material from the breast at the time of the primary diagnosis and who had developed a metastasis at any time during the subsequent course. For these patients, tumor and patient characteristics were used to determine prognostic factors relative to overall survival after the diagnosis of distant metastases. Ki-67 was added to this model to investigate whether this might improve the prediction of overall survival. In the multivariate Cox model, age at diagnosis, body mass index, nodal status, tumor size, ER and PR status, and time from diagnosis to metastasis were identified as relevant prognostic factors. Adding Ki-67 to the model improved the prediction of overall survival. There was also a significant and relevant interaction with the PR status. In patients with a low-proliferation primary tumor, a high level of PR expression would indicate an extraordinarily good prognosis (HR 0.39; 95 % CI, 0.23-0.66). In patients with higher-proliferation primary tumors, PR status was not capable of differentiating prognostic groups. Ki-67 is useful in addition to known prognostic factors for breast cancer. It is able to indicate a group of women with a poorer prognosis, specifically in the group of patients with PR-positive breast cancer.

  10. Interplay between binding affinity and kinetics in protein-protein interactions.

    PubMed

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. PMID:27018856

  11. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    PubMed

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  12. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  13. Interplay between binding affinity and kinetics in protein-protein interactions.

    PubMed

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc.

  14. Electron Affinities, Fluoride Affinities, and Heats of Formation of the Second Row Transition Metal Hexafluorides: MF6 (M = Mo, Tc, Ru, Rh, Pd, Ag)

    SciTech Connect

    Craciun, Raluca; Long, Rebecca T.; Dixon, David A.; Christe, Karl O.

    2010-07-22

    High-level electronic structure calculations were used to evaluate reliable, self-consistent thermochemical data sets for the second row transition metal hexafluorides. The electron affinities, heats of formation, first (MF{sub 6} {yields} MF{sub 5} + F) and average M-F bond dissociation energies, and fluoride affinities of MF{sub 6} (MF{sub 6} + F{sup -} {yields} MF{sub 7}{sup -}) and MF{sub 5} (MF{sub 5} + F{sup -} {yields} MF{sub 6}{sup -}) were calculated. The electron affinities are higher than those of the corresponding third row hexafluorides, making them stronger one-electron oxidizers. The calculated electron affinities, in good agreement with the available experimental values, are 4.23 eV for MoF{sub 6}, 5.89 eV for TcF{sub 6}, 7.01 eV for RuF{sub 6}, 6.80 eV for RhF{sub 6}, 7.95 eV for PdF{sub 6}, and 8.89 eV for AgF{sub 6}. The corresponding pentafluorides are also very strong Lewis acids, although their acidities on the pF{sup -} scale are about one unit lower than those of the third row pentafluorides. The performance of a wide range of DFT exchange-correlation functionals was benchmarked by comparing them to our more accurate CCSD(T) results.

  15. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

    Among the essential steps to be taken in structural health monitoring systems, damage prognosis would be the field that is least investigated due to the complexity of the uncertainties. This paper presents the possibility of using Affine Arithmetic for uncertainty propagation of crack damage in damage prognosis. The structures examined are thin rectangular plates made of titanium alloys with central mode I cracks and a composite plate with an internal delamination caused by mixed mode I and II fracture modes, under a harmonic uniaxial loading condition. The model-based method for crack growth rates are considered using the Paris Erdogan law model for the isotropic plates and the delamination growth law model proposed by Kardomateas for the composite plate. The parameters for both models are randomly taken and their uncertainties are considered as defined by an interval instead of a probability distribution. A Monte Carlo method is also applied to check whether Affine Arithmetic (AA) leads to tight bounds on the lifetime of the structure.

  16. High-affinity Cyclic Peptide Matriptase Inhibitors*

    PubMed Central

    Quimbar, Pedro; Malik, Uru; Sommerhoff, Christian P.; Kaas, Quentin; Chan, Lai Y.; Huang, Yen-Hua; Grundhuber, Maresa; Dunse, Kerry; Craik, David J.; Anderson, Marilyn A.; Daly, Norelle L.

    2013-01-01

    The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase. PMID:23548907

  17. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  18. Affine conformal vectors in space-time

    NASA Astrophysics Data System (ADS)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  19. Feature-enhancing zoom to facilitate Ki-67 hot spot detection

    NASA Astrophysics Data System (ADS)

    Molin, Jesper; Shaga Devan, Kavitha; Wârdell, Karin; Lundström, Claes

    2014-03-01

    Image processing algorithms in pathology commonly include automated decision points such as classifications. While this enables efficient automation, there is also a risk that errors are induced. A different paradigm is to use image processing for enhancements without introducing explicit classifications. Such enhancements can help pathologists to increase efficiency without sacrificing accuracy. In our work, this paradigm has been applied to Ki-67 hot spot detection. Ki-67 scoring is a routine analysis to quantify the proliferation rate of tumor cells. Cell counting in the hot spot, the region of highest concentration of positive tumor cells, is a method increasingly used in clinical routine. An obstacle for this method is that while hot spot selection is a task suitable for low magnification, high magnification is needed to discern positive nuclei, thus the pathologist must perform many zooming operations. We propose to address this issue by an image processing method that increases the visibility of the positive nuclei at low magnification levels. This tool displays the modified version at low magnification, while gradually blending into the original image at high magnification. The tool was evaluated in a feasibility study with four pathologists targeting routine clinical use. In a task to compare hot spot concentrations, the average accuracy was 75+/-4.1% using the tool and 69+/-4.6% without it (n=4). Feedback on the system, gathered from an observer study, indicate that the pathologists found the tool useful and fitting in their existing diagnostic process. The pathologists judged the tool to be feasible for implementation in clinical routine.

  20. Thermal chemistry of the Cu-KI5 atomic layer deposition precursor on a copper surface

    SciTech Connect

    Ma, Qiang; Zaera, Francisco

    2015-01-01

    The thermal chemistry of a Cu(I) ketoiminate complex, Cu-KI5, resulting from the modification of the known Air Products CupraSelect{sup ®} copper CVD precursor Cu(hfac)(tmvs) designed to tether the two ligands via an isopropoxide linker, was studied under ultrahigh vacuum on a Cu(110) single-crystal surface by using a combination of temperature programmed desorption (TPD) and x-ray photoelectron spectroscopy. Adsorption at low temperatures was determined to take place via the displacement of the vinyl ligand by the surface. Molecular desorption was seen at 210 K, and the evolution of Cu(II)-KI5{sub 2} was established to take place at 280 K, presumably from a disproportionation reaction that also leads to the deposition of Cu(0). Other sets of desorption products were seen at 150, 250, and 430 K, all containing copper atoms and small organic moieties with molecular masses below 100 amu. The latter TPD peak in particular indicates significant fragmentation of the ligands, likely at the C–N bond that holds the vinylsilane-isopropoxide moiety tethered to the ketoimine fragment, and possibly also at the union between the vinylsilane and the alkoxide linker. The 430 K temperature measured for this chemistry may set an upper limit for clean Cu film deposition, but since reactivity on the surface was also found to be inhibited at higher surface coverages, it may be delayed to higher temperatures under atomic layer deposition conditions.

  1. Immunohistochemical (Ki-67) study of endometrial maturation in mice after use of phosphodiesterase type 5 inhibitor

    PubMed Central

    Rashidi, Bahman; Rad, Jafar Soleimani; Rad, Leila Roshangar

    2015-01-01

    Background: Uterine receptivity for the implantation is a complicated process, that ovarian factors (hormonal), endometrium and embryo simultaneously are involved in this phenomenon. A successful implantation needs appropriate development of the endometrium. Furthermore, embryo must be capable of reacting with the endometrium and producing suitable adhesion molecules. This study aimed to examine one of endometrial maturation indices in mice before implantation, i.e., proliferation of stromal cells. Materials and Methods: A total of 40 adult female mice were divided into four groups: Control, gonadotropin, gonadotropin + progesterone, and gonadotropin + sildenafil citrate. The three experimental groups were first injected 7.5 IU of human chorionic gonadotropin (HCG) and then 7.5 IU of human menopausal gonadotropin (HMG). Then, every two female mice were placed in a cage with a male mouse for mating. Two groups were injected 1 mg of progesterone and 3 mg/kg of sildenafil citrate at intervals of 24, 48, and 72 h after injection of HMG. After 96 h, all the mice were killed, and their uterine samples subjected to tissue passage and prepared for analysis. Immunohistochemical method, Ki-67, and stromal mitotic cell count were used in this study. Results: Our observations in all groups showed changes in the luminal epithelium. ANOVA analysis Ki-67-positive stromal cells among all groups were not statistically significant. Conclusion: The results showed that administration of HMG and HCG following that of progesterone and sildenafil citrate could change the indices of endometrial maturation, and they were not involved in the phase immediately before implantation in stromal mitotic index. PMID:26380239

  2. Determination of proton affinities and acidity constants of sugars.

    PubMed

    Feng, Shuting; Bagia, Christina; Mpourmpakis, Giannis

    2013-06-20

    Proton transfer reactions play a key role in the conversion of biomass derived sugars to chemicals. In this study, we employ high level ab initio theoretical methods, in tandem with solvation effects to calculate the proton affinities (PA) and acidity constants (pKa) of various d-glucose and d-fructose tautomers (protonation-deprotonation processes). In addition, we compare the theoretically derived pH values of sugar solutions against experimentally measured pH values in our lab. Our results demonstrate that the protonation of any of the O atoms of the sugars is thermodynamically preferred without any significant variation in the PA values. Intramolecular hydrogen transfers, dehydration reactions, and ring-opening processes were observed, resulting from the protonation of specific hydroxyl groups on the sugars. Regarding the deprotonation processes (pKa), we found that the sugars' anomeric hydroxyls exhibit the highest acidity. The theoretically calculated pH values of sugar solutions are in excellent agreement with experimental pH measurements at low sugar concentrations. At higher sugar concentrations the calculations predict less acidic solutions than the experiments. In this case, we expect the sugars to act as solvents increasing the proton solvation energy and the acidity of the solutions. We demonstrated through linear relationships that the pKa values are correlated with the relative stability of the conjugate bases. The latter is related to hydrogen bonding and polarization of the C-O(-) bond. A plausible explanation for the good performance of the direct method in calculating the pKa values of sugars can be the presence of intramolecular hydrogen bonds on the conjugate base. Both theory and experiments manifest that fructose is a stronger acid than glucose, which is of significant importance in self-catalyzed biomass-relevant dehydration reactions. PMID:23706015

  3. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach. PMID:27498895

  4. Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity.

    PubMed Central

    Gennis, L S

    1976-01-01

    A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+.Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity. The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity. A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested. PMID:186779

  5. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

    PubMed Central

    Day, Christopher J.; Tran, Elizabeth N.; Semchenko, Evgeny A.; Tram, Greg; Hartley-Tassell, Lauren E.; Ng, Preston S. K.; King, Rebecca M.; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A.; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P.

    2015-01-01

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein–glycan or protein–protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host–glycan:bacterial–glycan pairs with equilibrium dissociation constants (KD) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  6. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

    PubMed

    Day, Christopher J; Tran, Elizabeth N; Semchenko, Evgeny A; Tram, Greg; Hartley-Tassell, Lauren E; Ng, Preston S K; King, Rebecca M; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P

    2015-12-29

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K(D)) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  7. Phosphatidylserine Reversibly Binds Cu2+ with Extremely High Affinity

    PubMed Central

    Monson, Christopher F.; Cong, Xiao; Robison, Aaron; Pace, Hudson P.; Liu, Chunming; Poyton, Matthew F.; Cremer, Paul S.

    2012-01-01

    Phosphatidylserine (PS) embedded within supported lipid bilayers (SLBs) was found to bind Cu2+ from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu2+ to PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85–90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu2+ concentrations and basic values pH (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion limited complex formation at basic pH, but rapid dissociation under acidic conditions. The tight binding of Cu2+ to PS may have physiological consequences under certain circumstances. PMID:22548290

  8. Automated detection of dual p16/Ki67 nuclear immunoreactivity in liquid-based Pap tests for improved cervical cancer risk stratification.

    PubMed

    Gertych, Arkadiusz; Joseph, Anika O; Walts, Ann E; Bose, Shikha

    2012-05-01

    The Papanicolau (Pap) test is a routine cytological procedure for early detection of dysplastic lesions in cervical epithelium. A reliable screening method is crucial for triage of women at risk; however manual screening and interpretation are associated with relatively low sensitivity and substantial interobserver diagnostic variability. P16 and Ki67 biomarkers have been recently proposed as adjunctive tools in the diagnosis of high-risk human papillomavirus (hrHPV) associated dysplasias to supplement the morphological characteristics of cells by additional colorimetric features. In this study, an automated technique for the evaluation of dual p16/Ki67 immunoreactivity in cervical cell nuclei is introduced. Smears stained with p16 and Ki67 antibodies were digitized, and analyzed by algorithms we developed. Gradient-based radial symmetry operator and adaptive processing of symmetry image were employed to obtain the nuclear mask. This step was followed by the extraction of features including pixel data and immunoreactivity signature from each nucleus. The features were analyzed by two support vector machine classifiers to assign a nucleus into one of four types of immunoreactivity: p16 positive (p16(+)/Ki67(-)), Ki67 positive (p16(-)/Ki67(+)), dual p16/Ki67 positive (p16(+)/Ki67(+)) and negative (p16(-)/Ki67(-)), respectively. Results obtained by our method correlated well with readings by two cytopathologists (n = 18,068 cells); p16(+)/Ki67(+) nuclei were classified with respective precisions of 77.1% and 82.6%. Specificity in identification of p16(-)/Ki67(-) nuclei was better than 99.5%, and the sensitivity in detection of all immunopositive nuclei was 86.3 and 89.4%, respectively. We found that the quantitative characterization of immunoreactivity provided by the additional highlighting of classified nuclei can positively impact the efficacy and screening outcome of the Pap test.

  9. Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54.

    PubMed

    Navarro, V M; Castellano, J M; Fernández-Fernández, R; Tovar, S; Roa, J; Mayen, A; Nogueiras, R; Vazquez, M J; Barreiro, M L; Magni, P; Aguilar, E; Dieguez, C; Pinilla, L; Tena-Sempere, M

    2005-01-01

    Loss-of-function mutations of the gene encoding GPR54, the putative receptor for the KiSS-1-derived peptide metastin, have been recently associated with hypogonadotropic hypogonadism, in both rodents and humans. Yet the actual role of the KiSS-1/GPR54 system in the neuroendocrine control of gonadotropin secretion remains largely unexplored. To initiate such analysis, the effects of KiSS-1 peptide on LH secretion were monitored using in vivo and in vitro settings under different experimental conditions. Central intracerebroventricular administration of KiSS-1 peptide potently elicited LH secretion in vivo over a range of doses from 10 pmol to 1 nmol. The effect of centrally injected KiSS-1 appeared to be mediated via the hypothalamic LHRH. However, no effect of central administration of KiSS-1 was detected on relative LHRH mRNA levels. Likewise, systemic (i.p. and i.v.) injection of KiSS-1 markedly stimulated LH secretion. This effect was similar in terms of maximum response to that of central administration of KiSS-1 and might be partially attributed to its ability to stimulate LH secretion directly at the pituitary. Finally, the LH-releasing activity of KiSS-1 was persistently observed after blockade of endogenous excitatory amino acid and nitric oxide pathways, i.e. relevant neurotransmitters in the neuroendocrine control of LH secretion. In summary, our results provide solid evidence for a potent stimulatory effect of KiSS-1 on LH release, acting at central levels (likely the hypothalamus) and eventually at the pituitary, and further document a novel role of the KiSS-1/GPR54 system as a relevant downstream element in the neuroendocrine network governing LH secretion. PMID:15375028

  10. Structure of a High-Affinity

    SciTech Connect

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps Res. Inst. /Simon Fraser U. /British Columbia U.

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  11. Interactions between CYP2E1 and CYP2B4: Effects on Affinity for NADPH-Cytochrome P450 Reductase and Substrate Metabolism

    PubMed Central

    Kenaan, Cesar; Shea, Erin V.; Lin, Hsia-lien; Zhang, Haoming; Pratt-Hyatt, Matthew J.

    2013-01-01

    Studies in microsomal and reconstituted systems have shown that the presence of one cytochrome P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence the catalytic activity of CYP2B4 under steady-state turnover conditions. The results show that CYP2E1 inhibits CYP2B4-mediated metabolism of benzphetamine (BNZ) with a Ki of 0.04 µM. However, CYP2B4 is not an inhibitor of CYP2E1-mediated p-nitrophenol hydroxylation. When these inhibition studies were performed with the artificial oxidant tert-butyl hydroperoxide, CYP2E1 did not significantly inhibit CYP2B4 activity. Determinations of the apparent KM and kcat of CYP2B4 for CPR in the presence of increasing concentrations of CYP2E1 revealed a mixed inhibition of CYP2B4 by CYP2E1. At low concentrations of CYP2E1, the apparent KM of CYP2B4 for CPR increased up to 23-fold with virtually no change in the kcat for the reaction, however, at higher concentrations of CYP2E1, the apparent KM of CYP2B4 for CPR decreased to levels similar to those observed in the absence of CYP2E1 and the kcat also decreased by 11-fold. Additionally, CYP2E1 increased the apparent KM of CYP2B4 for BNZ by 8-fold and the apparent KM did not decrease to its original value when saturating concentrations of CPR were used. While the individual apparent KM values of CYP2B4 and CYP2E1 for CPR are similar, the apparent KM of CYP2E1 for CPR in the presence of CYP2B4 decreased significantly, thus suggesting that CYP2B4 enhances the affinity of CYP2E1 for CPR and this may allow CYP2E1 to out-compete CYP2B4 for CPR. PMID:23043184

  12. Effectively nonlocal metric-affine gravity

    NASA Astrophysics Data System (ADS)

    Golovnev, Alexey; Koivisto, Tomi; Sandstad, Marit

    2016-03-01

    In metric-affine theories of gravity such as the C-theories, the spacetime connection is associated to a metric that is nontrivially related to the physical metric. In this article, such theories are rewritten in terms of a single metric, and it is shown that they can be recast as effectively nonlocal gravity. With some assumptions, known ghost-free theories with nonsingular and cosmologically interesting properties may be recovered. Relations between different formulations are analyzed at both perturbative and nonperturbative levels, taking carefully into account subtleties with boundary conditions in the presence of integral operators in the action, and equivalences between theories related by nonlocal redefinitions of the fields are verified at the level of equations of motion. This suggests a possible geometrical interpretation of nonlocal gravity as an emergent property of non-Riemannian spacetime structure.

  13. Affinity chromatography with an immobilized RNA enzyme.

    PubMed Central

    Vioque, A; Altman, S

    1986-01-01

    M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid. Images PMID:3526344

  14. Wetting on rough self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Palasantzas, George

    1995-05-01

    In this paper, we present a general investigation of the effective potential for complete wetting on self-affine rough surfaces. The roughness effect is investigated by means of the height-height correlation model in Fourier space ~(1+aξ2q2)-1-H. The parameters H and ξ are, respectively, the roughness exponent and the substrate in-plane correlation length. It is observed that the effect of H on the free interface profile is significant for ξ>ξ) regime is characterized by a power-law scaling ~Y-2.

  15. High-affinity neuropeptide Y receptor antagonists.

    PubMed Central

    Daniels, A J; Matthews, J E; Slepetis, R J; Jansen, M; Viveros, O H; Tadepalli, A; Harrington, W; Heyer, D; Landavazo, A; Leban, J J

    1995-01-01

    Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats. PMID:7568074

  16. Automatic gesture analysis using constant affine velocity.

    PubMed

    Cifuentes, Jenny; Boulanger, Pierre; Pham, Minh Tu; Moreau, Richard; Prieto, Flavio

    2014-01-01

    Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field. PMID:25570332

  17. Evaluation system of negative electron affinity photocathode

    NASA Astrophysics Data System (ADS)

    Fu, Rongguo; Chang, Benkang; Qian, Yunsheng; Wang, Guihua; Zong, Zhiyuan

    2001-10-01

    This article first describes the background of the research and manufacture of evaluation system of Negative Electron Affinity photocathode. This article designs a set of super high vacuum system for activating NEA photocathode on the base of activation theory, the process of design and debugging is given. The system is composed of three parts: super high vacuum system for GaAs material activation, multi-meter testing system, surface analysis system. The system is used for on-line evaluation of activating of NEA photocathode. The technical parameters and structure of the evaluation system of NEA photocathode are given in the paper. The system is finished and experiments are made. At last the picture of the system is given.

  18. Influence of experimental parameters on sonochemistry dosimetries: KI oxidation, Fricke reaction and H2O2 production.

    PubMed

    Merouani, Slimane; Hamdaoui, Oualid; Saoudi, Fethi; Chiha, Mahdi

    2010-06-15

    Central events of the ultrasonic action are the cavitation bubbles that can be considered as microreactors. Adiabatic collapse of cavitation bubbles leads to the formation of reactive species such as hydroxyl radicals (*OH), hydrogen peroxide (H(2)O(2)) and hydroperoxyl radicals (HOO*). Several chemical methods were used to detect the production of these reactive moieties in sonochemistry. In this work, the influence of several operational parameters on the sonochemistry dosimetries namely KI oxidation, Fricke reaction and H(2)O(2) production using 300 kHz ultrasound was investigated. The main experimental parameters showing significant effect in KI oxidation dosimetry were initial KI concentration, acoustic power and pH. The solution temperature showed restricted influence on KI oxidation. The acoustic power and liquid temperature highly affected Fricke reaction dosimetry. Operational conditions having important influence on H(2)O(2) formation were acoustic power, solution temperature and pH. For the three tested dosimetries, the sonochemical efficiency was independent of liquid volume. PMID:20211524

  19. Prognostic Significance of p27, Ki-67, and Topoisomerase lla Expression in Clinically Nonfunctioning Pancreatic Endocrine Tumors.

    PubMed

    Chang, Hee Jin; Batts, Kenneth P.; Lloyd, Ricardo V.; Sebo, Thomas J.; Thompson, Geoffrey B.; Lohse, Christine M.; Pankratz, Shane V.

    2000-01-01

    Nonfunctioning islet cell tumors or pancreatic endocrine tumors are the most common type of malignant islet cell tumor. Although previously detected usually at an advanced stage because of mass effect, the early detection rate of small localized disease has been increasing. To date it has been difficult to predict the clinical behavior in localized regional nonfunctioning tumors. To investigate potential markers predicting malignancy and poor prognosis in nonfunctioning pancreatic endocrine tumors, we analyzed the expression of Ki-67, topoisomerase Ila (Topolla), and p27, as well as a variety of clinicopathologic parameters in 76 cases of nonfunctioning islet cell tumors (23 benign cases and 53 malignant cases). Ki-67, Topolla. and p27 labeling indices were significantly different between benign and malignant tumors. Expression of Ki-67, Topolla, and p27 were associated with survival in patients with a malignant tumor in a univariate setting. However, only p27 and Topolla were jointly associated with survival in multivariate analysis. Immunohistochemical staining for p27, Topolla, and Ki-67 can be helpful in the diagnosis of nonfunctioning pancreatic endocrine tumor. Analysis of p27 and Topolla may also have potential utility as prognostic factors for malignant tumors.

  20. Polymorphisms rs12998 and rs5780218 in KiSS1 Suppressor Metastasis Gene in Mexican Patients with Breast Cancer

    PubMed Central

    Cruz Quevedo, Edhit Guadalupe; Mimendi Aguilar, Gabriela Monserrat; Juárez Aguilar, Luis Anselmo; Gutierrez Rubio, Susan Andrea; Flores Martínez, Silvia Esperanza; Dávalos Rodríguez, Ingrid Patricia; Sánchez Corona, José; Torres Morán, Martha Isabel; Rosales Gómez, Roberto Carlos; Morán Moguel, María Cristina

    2015-01-01

    Aims. KiSS1 is a metastasis suppressor gene associated with inhibition of cellular chemotaxis and invasion attenuating the metastasis in melanoma and breast cancer cell lines. Along the KiSS-1 gene at least 294 SNPs have been described; however the association of these polymorphisms as genetic markers for metastasis in breast cancer studies has not been investigated. Here we describe two simple PCR-RFLPs protocols to identify the rs5780218 (9DelT) and the rs12998 (E20K) KiSS1 polymorphisms and the allelic, genotypic, and haplotypic frequencies in Mexican general population (GP) and patients with benign breast disease (BBD) or breast cancer (BC). Results. The rs5780218 polymorphism was individually associated with breast cancer (P = 0.0332) and the rs12998 polymorphism shows statistically significant differences when GP versus case (BC and BBD) groups were compared (P < 0.0001). The H1 Haplotype (G/-) occurred more frequently in BC group (0.4256) whereas H2 haplotype (G/T) was the most prevalent in BBD group (0.4674). Conclusions. Our data indicated that the rs5780218 polymorphism individually confers susceptibility for development of breast cancer in Mexican population and a possible role as a genetic marker in breast cancer metastasis for H1 haplotype (Wt/variant) in KiSS1 gene must be analyzed in other populations. PMID:25810563

  1. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    SciTech Connect

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg . E-mail: jkobarg@lnls.br

    2006-03-17

    Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.

  2. ATRX, IDH1-R132H and Ki-67 immunohistochemistry as a classification scheme for astrocytic tumors

    PubMed Central

    Zhang, Wei; Wang, Guangzhi; Yao, Kun; Wang, Zhiliang; Li, Guanzhang; Qian, Zenghui; Li, Yongli; Jiang, Tao; Jiang, Chuanlu

    2016-01-01

    Recurrence and progression to higher grade lesions are key biological events and characteristic behaviors in the evolution process of glioma. Malignant astrocytic tumors such as glioblastoma (GBM) are the most lethal intracranial tumors. However, the clinical practicability and significance of molecular parameters for the diagnostic and prognostic prediction of astrocytic tumors is still limited. In this study, we detected ATRX, IDH1-R132H and Ki-67 by immunohistochemistry and observed the association of IDH1-R132H with ATRX and Ki-67 expression. There was a strong association between ATRX loss and IDH1-R132H (p<0.0001). However, Ki-67 high expression restricted in the tumors with IDH1-R132H negative (p=0.0129). Patients with IDH1-R132H positive or ATRX loss astrocytic tumors had a longer progressive- free survival (p<0.0001, p=0.0044, respectively). High Ki-67 expression was associated with shorter PFS in patients with astrocytic tumors (p=0.002). Then we characterized three prognostic subgroups of astrocytic tumors (referred to as A1, A2 and A3). The new model demonstrated a remarkable separation of the progression interval in the three molecular subgroups and the distribution of patients’ age in the A1-A2-A3 model was also significant different. This model will aid predicting the overall survival and progressive time of astrocytic tumors’ patients. PMID:27713914

  3. Prognostic implications of p53 protein, epidermal growth factor receptor, and Ki-67 labelling in brain tumours.

    PubMed Central

    Jaros, E.; Perry, R. H.; Adam, L.; Kelly, P. J.; Crawford, P. J.; Kalbag, R. M.; Mendelow, A. D.; Sengupta, R. P.; Pearson, A. D.

    1992-01-01

    The expression of p53 protein, epidermal growth factor receptor (EGFR), and Ki-67 nuclear antigen was examined by immunohistochemistry in biopsies of 16 types of human brain tumours, including 43 astrocytomas. P53 protein, almost certainly its mutant form, was expressed in seven of the 16, and EGFR in 11 of the 16 types of tumours. In astrocytomas both the proportion of tumours which expressed p53 or EGFR increased with grade of malignancy as did the mean Ki-67 labelling index (LI): p53-0% in grade 1, 17% in grade 2, 38% in grade 3, 65% in grade 4; EGFR-0% in grade 1, 33% in grade 2, 85% in grade 3, 95% in grade 4; mean Ki-67 L1-1.1% in grades 1 and 2, 8.3% in grade 3, and 13.4% in grade 4. Astrocytomas which expressed p53 or EGFR had a significantly higher Ki-67 LI at P less than 0.05 (11.8% and 10.7%, resp.) than those that did not (6.2% or 4.1%, resp.). Patients with astrocytomas expressing p53 or EGFR had a significantly reduced survival (P = 0.035 and P = 0.007, resp.): only 11% of the p53 + ve and 13% of the EGFR + ve patients were alive at 100 weeks following diagnosis compared to 36% of p53-ve or 60% of EGFR-ve patients. Patients with Ki-67 LI greater than 5% had a reduced survival (P less than 0.0001)--none survived beyond 86 weeks following diagnosis, whilst 63% of patients with less than 5% positive cells were still alive at 100 weeks. The univariate analysis showed that in astrocytomas expression of p53 mutants, EGFR protein, and Ki-67 greater than 5% are associated with malignant progression and poor prognosis. The multivariate analysis revealed that only tumour grade and Ki-67LI were independent prognostic factors for survival. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 9 PMID:1503912

  4. p16(INK4a) /Ki-67 co-expression specifically identifies transformed cells in the head and neck region.

    PubMed

    Prigge, Elena-Sophie; Toth, Csaba; Dyckhoff, Gerhard; Wagner, Steffen; Müller, Franziska; Wittekindt, Claus; Freier, Kolja; Plinkert, Peter; Hoffmann, Jürgen; Vinokurova, Svetlana; Klussmann, Jens Peter; von Knebel Doeberitz, Magnus; Reuschenbach, Miriam

    2015-04-01

    p16(INK4a) immunohistochemical overexpression is an overall reliable surrogate marker of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC). However, cases of ambiguous p16(INK4a) overexpression are regularly detected in the head and neck: p16(INK4a) expression can be observed in non-malignant tissue, such as tonsillar crypt epithelium and a proportion of branchial cleft cysts. Additionally, diverse patterns of p16(INK4) expression can complicate interpretation of "p16(INK4a) -positivity". These aspects impede the unrestricted application of p16(INK4a) as a diagnostic marker in the head and neck. We hypothesized that combined detection of p16(INK4a) and the proliferation marker Ki-67 could support clarification of ambiguous p16(INK4a) expression in the head and neck by specifically indicating p16(INK4a) -expressing cells with proliferative activity. p16(INK4a) /Ki-67 co-expression in a combined staining procedure was correlated to distinct p16(INK4a) expression patterns and HPV status (HPV DNA followed by E6*I oncogene mRNA detection) in 147 HNSCC and 50 non-malignant head and neck samples. p16(INK4a) /Ki-67 co-expression only occurred in transformed cells of the head and neck. Co-expression was never detected in non-transformed cells. Combined p16(INK4a) /Ki-67 expression was stringently associated with a diffuse p16(INK4a) expression pattern. All HPV oncogene-expressing HNSCC showed p16(INK4a) /Ki-67 co-expression. We demonstrate that p16(INK4a) /Ki-67 co-expression occurs exclusively in transformed cells of the head and neck. Our findings indicate a substantial impact of combined p16(INK4a) /Ki-67 expression in the assessment of ambiguous p16(INK4a) expression in the head and neck by specifically identifying p16(INK4a) -expressing cells with proliferative activity. This property will be of considerable significance for head and neck histo- and cytopathology.

  5. Evaluation of cervical cone biopsies for coexpression of p16INK4a and Ki-67 in epithelial cells.

    PubMed

    Reuschenbach, Miriam; Seiz, Mirjam; von Knebel Doeberitz, Christina; Vinokurova, Svetlana; Duwe, Alexander; Ridder, Ruediger; Sartor, Heike; Kommoss, Friedrich; Schmidt, Dietmar; von Knebel Doeberitz, Magnus

    2012-01-15

    Diffuse overexpression of p16(INK4a) in basal and parabasal cells of cervical epithelium is a hallmark of human papillomavirus-mediated transformation. Focal p16(INK4a) expression is occasionally observed in nondysplastic epithelium. In normal cells, expression of p16(INK4a) triggers cell cycle arrest. However, cells undergoing transformation in intraepithelial lesions actively proliferate. To prove that the different expression patterns of p16(INK4a) , i.e., focal versus diffuse, reflect biologically different entities, we hypothesized that p16(INK4a) -positive cells in epithelia displaying focal p16(INK4a) expression pattern do not coexpress proliferation-associated Ki-67 protein, while p16(INK4a) -positive cells in lesions with diffuse p16(INK4a) expression may do. A total of 138 cervical cone biopsies were stained for the expression of p16(INK4a) and Ki-67 using a primary antibody cocktail. All metaplastic lesions (n = 21) displayed focal staining for p16(INK4a) , and in all of these lesions p16(INK4a) -positive cells were found to be negative for Ki-67 expression. Diffuse expression of p16(INK4a) was observed in 12/21 (57.1%) cervical intraepithelial neoplasia (CIN) 1 lesions, all of them simultaneously showed Ki-67 immunoreactivity in a large proportion of p16(INK4a) -positive cells. Seventeen of 23 (73.9%) CIN2 lesions and all 27 (100%) CIN3/carcinoma in situ (CIS) as well as all 46 (100%) carcinoma cases displayed diffuse and combined expression of p16(INK4a) and Ki-67. Coexpression of Ki-67 and p16(INK4a) in the same cell is entirely restricted to cervical lesions displaying diffuse p16(INK4a) expression, whereas in lesions with focal p16(INK4a) expression, p16(INK4a) -expressing cells are negative for Ki-67. Thus, diffuse expression of p16(INK4a) reflects lesions with proliferation-competent cells, while p16(INK4a) -expressing cells associated with focal expression patterns are cell cycle arrested.

  6. Purification of glycolytic enzymes by using affinity-elution chromatography.

    PubMed Central

    Scopes, R K

    1977-01-01

    1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures. PMID:192194

  7. Antibody-based affinity cryo-EM grid.

    PubMed

    Yu, Guimei; Li, Kunpeng; Jiang, Wen

    2016-05-01

    The Affinity Grid technique combines sample purification and cryo-Electron Microscopy (cryo-EM) grid preparation into a single step. Several types of affinity surfaces, including functionalized lipids monolayers, streptavidin 2D crystals, and covalently functionalized carbon surfaces have been reported. More recently, we presented a new affinity cryo-EM approach, cryo-SPIEM, which applies the traditional Solid Phase Immune Electron Microscopy (SPIEM) technique to cryo-EM. This approach significantly simplifies the preparation of affinity grids and directly works with native macromolecular complexes without need of target modifications. With wide availability of high affinity and high specificity antibodies, the antibody-based affinity grid would enable cryo-EM studies of the native samples directly from cell cultures, targets of low abundance, and unstable or short-lived intermediate states.

  8. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  9. Concurrent low- and high-affinity sulfate reduction kinetics in marine sediment

    NASA Astrophysics Data System (ADS)

    Harder Tarpgaard, Irene; Røy, Hans; Jørgensen, Bo Barker

    Bacterial sulfate reduction in marine sediments generally occurs in the presence of high millimolar concentrations of sulfate. Published data indicate that low sulfate concentrations may limit sulfate reduction rates below 0.2-2 mM. Yet, high sulfate reduction rates occur in the 1-100 μM range in freshwater sediments and at the sulfate-methane transition in marine sediments. Through a combination of 35S-tracer experiments, including initial velocity experiments and time course experiments, we searched for different sulfate affinities in the mixed community of sulfate reducers in a marine sediment. We supported the radiotracer experiments with a highly sensitive ion chromatographic technique for sulfate with a detection limit of 0.15 μM SO 42- in marine pore water. Our results showed that high and low affinities for sulfate co-occur and that the applied experimental approach may determine the observed apparent half saturation constant, Km. Our experimental and model data both show that sulfate reduction in the studied marine sediment could be explained by two dominating affinities for sulfate: a low affinity with a mean half saturation constant, Km, of 430 μM SO 42- and a high affinity with a mean Km of 2.6 μM SO 42-. The high-affinity sulfate reduction was thermodynamically un-constrained down to <1 μM SO 42-, both in our experiments and under in situ conditions. The reduction of radio-labeled sulfate was partly reversible due to concurrent re-oxidation of sulfide by Fe(III) and possibly due to a reversibility of the enzymatic pathway of sulfate reduction. A literature survey of apparent Km values for sediments and pure cultures is presented and discussed.

  10. p53 is not related to Ki-67 immunostaining in the epithelial and mesenchymal components of female genital tract carcinosarcomas.

    PubMed

    Bałon, Beata; Kaznowska, Ewa; Ignatov, Atanas; Steć, Anna; Semczuk-Sikora, Anna; Schneider-Stock, Regine; Jóźwik, Maciej; Sulkowski, Stanisław; Cybulski, Marek; Szumiło, Justyna; Semczuk, Andrzej

    2013-10-01

    Carcinosarcomas (CSs) are composed of two separate histological components and are rare neoplasms of the female genital tract. Therefore, CS pathogenesis has not yet been fully elucidated. In the present study, immunohistochemical techniques were used to determine the role of p53 and Ki-67 overexpression in female genital tract CSs. The study group was comprised of 36 patients with CSs originating from the uterus (n=31), cervix (n=3) and ovary (n=2), as well as 3 metastatic tissues. p53 was overexpressed in the epithelial component of 23 out of 36 (64%) tumors, and in the mesenchymal component of 20 out of 36 (56%) tumors. In both CS components, there was a significant correlation between p53 overexpression and patient age and ovarian metastases. Ki-67 overexpression was detected in the epithelial component in 15 out of 36 (42%) cases, and in the mesenchymal component in 13 out of 36 (36%) neoplasms. There was a significant correlation of p53 overexpression between the carcinomatous and sarcomatous components (R=0.884, P<0.001). A significant correlation was also found in Ki-67 immunoreactivity between the two CS components (R=0.676, P<0.001). However, p53 overexpression was not correlated with Ki-67 immunostaining in both tumor components. In conclusion, based on immunohistochemical results, p53 was overexpressed in more than half of the female genital tract CSs included in the present study, either at the epithelial or mesenchymal component. The correlation between p53 or Ki-67 overexpression in both tumor components supports the combination theory of histogenesis in the majority of these tumors.

  11. Low power laser irradiation stimulates cell proliferation via proliferating cell nuclear antigen and Ki-67 expression during tissue repair

    NASA Astrophysics Data System (ADS)

    Prabhu, Vijendra; Rao, Bola Sadashiva Satish; Mahato, Krishna Kishore

    2015-03-01

    Low power laser irradiation (LPLI) is becoming an increasingly popular and fast growing therapeutic modality in dermatology to treat various ailments without any reported side effects. In the present study an attempt was made to investigate the proliferative potential of red laser light during tissue repair in Swiss albino mice. To this end, full thickness excisional wounds of diameter 15 mm created on mice were exposed to single dose of Helium-Neon laser (632.8 nm; 7 mW; 4.02 mWcm-2; Linear polarization) at 2 Jcm-2 and 10 Jcm-2 along with un-illuminated controls. The granulation tissues from all the respective experimental groups were harvested on day 10 post-wounding following euthanization. Subsequently, tissue regeneration potential of these laser doses under study were evaluated by monitoring proliferating cell nuclear antigen and Ki-67 following the laser treatment and comparing it with the un-illuminated controls. The percentages of Ki-67 or PCNA positive cells were determined by counting positive nuclei (Ki-67/PCNA) and total nuclei in five random fields per tissue sections. Animal wounds treated with single exposure of the 2 Jcm-2 indicated significant elevation in PCNA (P<0.01) and Ki-67 (P<0.05 compared to un-illuminated control and P<0.01 compared to 10 Jcm-2) expression as compared to other tested experimental groups as evidenced by the microscopy results in the study. In summary, the findings of the present study have clearly demonstrated the regulation of cell proliferation by LPLI via PCNA and Ki-67 expression during tissue regeneration.

  12. Effect of KI/AgI on the thermal and optical properties of the GeS₂-In₂S₃ chalcogenide glasses.

    PubMed

    Wang, Guoxiang; Nie, Qiuhua; Wang, Xunsi; Chen, Fen; Dai, Shixun; Xu, Tiefeng; Shen, Xiang; Wang, Dagang; Lv, Xin

    2010-11-01

    GeS₂-In₂S₃-KI/AgI chalcohalide glasses were prepared by traditional melt-quenching method and the glass-forming region was determined. The dependence of glass properties on composition as formula of (100 - x)(0.75GeS₂-0.25In₂S₃) - x(In₂S₃-KI/AgI) was investigated. The allowed indirect transition of samples was calculated according to the classical Tauc equation. The results show that 50 mol% AgI can be introduced in the glassy matrix while only 40 mol% of KI can be incorporated in the GeS₂-In₂S₃-KI glass system. GeS₂-In₂ S₃-AgI glass system has larger density, higher refractive index and better thermal stability, while GeS₂-In₂S₃-KI glass system has shorter visible cut-off wavelength and higher optical band gap. PMID:20800537

  13. Affinity chromatography for purification of two urokinases from human urine.

    PubMed

    Takahashi, R; Akiba, K; Koike, M; Noguchi, T; Ezure, Y

    2000-05-26

    A new affinity chromatography (hydrophobic-mediated affinity chromatography), which was characterized by the matrix having both affinity site to urokinase and hydrophobic site, was established for the purification of urokinase from human urine. The hydrophobic affinity matrix (tentatively named PAS in the text) was prepared by immobilizing 6-aminocaproic acid on Sepharose CL-6B, followed by a coupling p-aminobenzamidine to a part of the hydrophobic site on the matrix. The PAS matrix was applied to the purification of urokinase from human urine, and high- and low-molecular weight pure urokinases were efficiently obtained in high yield by the present method. PMID:10892585

  14. Three-dimensional site response at KiK-net downhole arrays

    USGS Publications Warehouse

    Thompson, Eric M.; Tanaka, Yasuo; Baise, Laurie G.; Kayen, Robert E.

    2010-01-01

    Ground motions at two Kiban-Kyoshin Network (KiK-net) strong motion downhole array sites in Hokkaido, Japan (TKCH08 in Taiki and TKCH05 in Honbetsu) illustrate the importance of three-dimensional (3D) site effects. These sites recorded the M8.0 2003 Tokachi-Oki earthquake, with recorded accelerations above 0.4 g at both sites as well as numerous ground motions from smaller events. Weak ground motions indicate that site TKCH08 is well modeled with the assumption of plane SH waves traveling through a 1D medium (SH1D), while TKCH05 is characteristic of a poor fit to the SH1D theoretical response. We hypothesized that the misfit at TKCH05results from the heterogeneity of the subsurface. To test this hypothesis, we measured four S-wave velocity profiles in the vicinity (< 300 m) of each site with the spectral analysis of surface waves (SASW) method. This KiK-net site pair is ideal for assessing the relative importance of 3D site effects and nonlinear site effects. The linear ground motions at TKCH05 isolate the 3D site effects, as we hypothesized from the linear ground motions and confirmed with our subsequent SASW surveys. The Tokachi-Oki time history at TKCH08 isolates the effects of nonlinearity from spatial heterogeneity because the 3D effects are negligible. The Tokachi-Oki time history at TKCH05 includes both nonlinear and 3D site effects. Comparisons of the accuracy of the SH1D model predictions of these surface time histories from the downhole time histories indicates that the 3D site effects are at least as important as nonlinear effects in this case. The errors associated with the assumption of a 1D medium and 1D wave propagation will be carried into a nonlinear analysis that relies on these same assumptions. Thus, the presence of 3D effects should be ruled out prior to a 1D nonlinear analysis. The SH1D residuals show that 3D effects can be mistaken for nonlinear effects.

  15. Ki-67 Is an Independent Predictor of Metastasis and Cause-Specific Mortality for Prostate Cancer Patients Treated on Radiation Therapy Oncology Group (RTOG) 94-08

    SciTech Connect

    Verhoven, Bret; Yan, Yan; Ritter, Mark; Khor, Li-Yan; Hammond, Elizabeth; Jones, Christopher; Amin, Mahul; Bahary, Jean-Paul; Zeitzer, Kenneth; Pollack, Alan

    2013-06-01

    Purpose: The association of Ki-67 staining index (Ki67-SI) with overall survival (OS), disease-specific mortality (DSM), distant metastasis (DM), and biochemical failure (BF) was examined in men with favorable- to intermediate-risk prostate cancer receiving radiation therapy (RT) alone or with short-term androgen deprivation (ADT) in Radiation Therapy Oncology Group (RTOG) 94-08. Methods and Materials: 468 patients (23.6%) on RTOG 94-08 had sufficient tissue for Ki67-SI analysis. The median follow-up time was 7.9 years. Ki67-SI was determined by immunohistochemistry and quantified manually and by image analysis. Correlative analysis versus clinical outcome was performed using the third quartile (≥Q3) cutpoint. A proportional hazards multivariable analysis (MVA) dichotomized covariates in accordance with trial stratification and randomization criteria. Results: In MVAs adjusted for all treatment covariates, high Ki67-SI (≥Q3) was correlated with increased DSM (hazard ratio [HR] 2.48, P=.03), DM (HR 3.5, P=.002), and BF (HR 3.55, P<.0001). MVA revealed similar Ki67-associated hazard ratios in each separate treatment arm for DSM, DM, and BF; these reached significance only for DM in the RT-alone arm and for BF in both arms. Ki67-SI was not a significant predictor of intraprostatic recurrence assessed by repeated biopsy 2 years after treatment. Patients with a high or low Ki67-SI seemed to experience a similar relative benefit from the addition of ADT to radiation. Conclusions: High Ki67-SI independently predicts for increased DSM, DM, and protocol BF in primarily intermediate-risk prostate cancer patients treated with RT with or without ADT on RTOG 94-08 but does not predict for local recurrence or for increased relative benefit from ADT. This and prior studies lend support for the use of Ki67-SI as a stratification factor in future trials.

  16. Differential Ovarian Expression of KiSS-1 and GPR-54 During the Estrous Cycle and Photoperiod Induced Recrudescence in Siberian Hamsters (Phodopus sungorus)

    PubMed Central

    Shahed, Asha; Young, Kelly A.

    2008-01-01

    Kisspeptins, coded by the KiSS-1 gene, regulate aspects of the reproductive axis by stimulating GnRH release via the G protein coupled receptor, GPR54. Recent reports show that KiSS/GPR54 may be key mediators in photoperiod-controlled reproduction in seasonal breeders, and that KiSS-1/GPR54 are expressed in the hypothalamus, ovaries, placenta, and pancreas. This study examined the expression of KiSS-1/GPR54 mRNA and protein in ovaries of Siberian hamsters (Phodopus sungorus). Ovaries from cycling hamsters were collected during proestrus (P), estrus (E), diestrus I (DI), and diestrus II (DII). To examine KiSS-1/GPR54 during stimulated recrudescence, additional hamsters were maintained either in long day (LD 16L:8D, control) or short day (SD 8L:16D) for 14 weeks and then transferred to LD for 0–8 weeks. Staining of KiSS-1/GPR54 protein was detected by immunohistochemistry in steroidogenic cells of preantral and antral follicles, and corpora lutea. Immunostaining peaked in P and E, but decreased in the diestrus stages (p<0.05). In recrudescing ovaries, KiSS-1/GPR54 immunostaining was low after 14 wks of SD exposure (post transfer [PT] wk0), and increased during the early weeks of recrudescence. Expression of KiSS-1/GPR54 mRNA was low with short day exposure, but increased during recrudescence and was higher at PT wk8 as compared to PTwks 0 and 2 (p<0.05). The elevated KiSS-1/ GPR54 expression during P and E suggests a potential role in ovulation in Siberian hamsters. Transient increases in KiSS-1/GPR54 expression following LD stimulation are also suggestive of possible involvement in ovulation and/or restoration of ovarian function. PMID:18937338

  17. Studies on recombinant single chain Jacalin lectin reveal reduced affinity for saccharides despite normal folding like native Jacalin.

    PubMed

    Sahasrabuddhe, Anagh A; Gaikwad, Sushama M; Krishnasastry, M V; Khan, M Islam

    2004-12-01

    Sugar binding studies, inactivation, unfolding, and refolding of native Jacalin (nJacalin) from Artocarpus integrifolia and recombinant single-chain Jacalin (rJacalin) expressed in Escherichia coli were studied by intrinsic fluorescence and thermal and chemical denaturation approaches. Interestingly, rJacalin does not undergo any proteolytic processing in an E. coli environment. It has 100fold less affinity for methyl-alpha-galactose (Ka: 2.48 x 10(2)) in comparison to nJacalin (Ka: 1.58 x 10(4)), and it also binds Thomsen-Friedenreich (TF) disaccharide (Galbeta1-3GalNAc) with less affinity. Overall sugar binding characteristics of rJacalin are qualitatively similar to that of nJacalin (GalKI, and CsCl indicated that the tryptophan residues have full accessibility to the neutral quencher and poor accessibility to ionic quenchers. In summary, biophysical and biochemical studies on the native versus recombinant Jacalin suggest that post-translational modification, i.e., the processing of Jacalin into two chains is probably not a prerequisite for sugar binding but may be required for higher affinity.

  18. Complex Affine Toda Theories and Soliton Solutions

    NASA Astrophysics Data System (ADS)

    Zhu, Zhiqing

    1995-01-01

    Toda field theories (TFT's) constitute a large class of integrable (1 + 1)-dimensional field theories that are relativistically invariant: included are conformal field theories and integrable deformations away from conformality. Because they are soluble, for example, by the inverse scattering method, and because they are related to many other areas of field theory, they have been studied extensively in recent years. Hirota's method is a straightforward procedure to obtain soliton solutions to non-linear integrable equations. In Hirota's method, one first writes the nonlinear equations in Hirota's bilinear form, and then expands the so called tau-functions as a power series in an arbitrary parameter. The power series terminates at some finite order, thus the solutions obtained are exact. For an N-soliton solution, the number of terms in the expansion grows exponentially with N, making direct calculation of N-soliton solutions difficult. We extend Hirota's one -parameter expansion to an N-parameter expansion. In the new expansion series, many terms are identical to those in the (N - 1)-soliton solutions, and new terms grow only linearly with N. Furthermore, we note that the expansion must terminate at some finite order, thus the vanishing of higher order terms can be used as constraints on these new terms. It turns out that these constraints can be used to determine the new terms completely. We used this extended Hirota's method to find N-soliton solutions for complex affine TFT's based on a simply-laced Kac-Moody algebra. Soliton solutions for non-simply-laced complex ATFT's can be obtained for those of simply-laced complex ATFT's by folding or twisting. Even though some soliton solutions have already been obtained for complex ATFT's by various methods, the physical implications of these solutions have not yet been thoroughly discussed. There are infinitely many distinct topological solitons in any given complex affine Toda field theory and most of them have complex

  19. Associations of teacher credibility and teacher affinity with learning outcomes in health classrooms

    PubMed Central

    Anderman, Eric M.; O’Connell, Ann A.

    2011-01-01

    In the present study (N = 633), we examine the role of teacher credibility and teacher affinity in classrooms. We explore the relations among these two characteristics and student gains in knowledge and valuing of learning about HIV and pregnancy prevention across high school classrooms. Results marshaled support for the notion that teacher characteristics are associated with classroom-level gains in learning outcomes. Above and beyond student-level predictors, teacher credibility (aggregated to the classroom level) was positively related to increases in knowledge across classrooms, whereas aggregated teacher affinity was positively related to an increased valuing of learning about HIV and pregnancy prevention across classrooms. Future directions and implications for practice are discussed. PMID:24876800

  20. WU and KI Polyomaviruses in Respiratory Samples from Allogeneic Hematopoietic Cell Transplant Recipients

    PubMed Central

    Campbell, Angela P.; Guthrie, Katherine A.; Wright, Nancy L.; Englund, Janet A.; Corey, Lawrence; Boeckh, Michael

    2012-01-01

    Data are limited regarding 2 new human polyomaviruses, KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV), in immunocompromised patients. We used real-time PCR to test for these and 12 respiratory viruses in 2,732 nasal wash samples collected during the first year after allogeneic hematopoietic cell transplantation from 222 patients. Specimens were collected weekly until day 100; then at least every 3 months. One year after hematopoietic cell transplantation, the cumulative incidence estimate was 26% for KIPyV and 8% for WUPyV. Age <20 years predicted detection of KIPyV (hazard ratio [HR] 4.6) and WUPyV (HR 4.4), and detection of a respiratory virus in the previous 2 weeks predicted KIPyV detection (HR 3.4). Sputum production and wheezing were associated with detection of KIPyV in the past week and WUPyV in the past month. There were no associations with polyomavirus detection and acute graft versus host disease, cytomegalovirus reactivation, neutropenia, lymphopenia, hospitalization, or death. PMID:23017213

  1. Phase transitions and relaxation dynamics in (NH 4I) x(KI) 1- x mixed crystals

    NASA Astrophysics Data System (ADS)

    Güthoff, F.; Ohl, M.; Reehuis, M.; Loidl, A.

    1999-06-01

    Dielectric studies, as well as elastic and quasielastic neutron scattering experiments were performed on the mixed molecular system (NH 4I) x(KI) 1- x for ammonium concentrations 0.55⩽ x⩽0.8. Based on these experiments we present a detailed and improved phase diagram, which reveals a variety of different structural phases with different degrees of orientational order, as well as different glass states with frozen-in orientational disorder. Special attention has been devoted to the recently discovered ε-phase which is characterized by the appearance of a superstructure reflection at the (3 0 0) reciprocal lattice point. In constant- Q scans at zero energy transfer we followed the temperature dependencies of these superstructure reflections and determined correlation lengths for all concentrations investigated. We found short-range order of the ε-type structure for x=0.55, while long-range order has been observed for all other concentrations. In addition, we performed constant- Q scans at the (3 0 0) reciprocal lattice point for several temperatures. From a purely phenomenological analysis, two time scales were observed which we attributed (i) to structural relaxations at the phase transition from the cubic high-temperature to the trigonal low-temperature ε-phase and (ii) to orientational relaxations of the NH 4+-tetrahedra in a slightly distorted environment. This interpretation is in accordance with the dielectric results which also provide experimental evidence for the appearance of two time scales.

  2. High Energy Density Aqueous Electrochemical Capacitors with a KI-KOH Electrolyte.

    PubMed

    Wang, Xingfeng; Chandrabose, Raghu S; Chun, Sang-Eun; Zhang, Tianqi; Evanko, Brian; Jian, Zelang; Boettcher, Shannon W; Stucky, Galen D; Ji, Xiulei

    2015-09-16

    We report a new electrochemical capacitor with an aqueous KI-KOH electrolyte that exhibits a higher specific energy and power than the state-of-the-art nonaqueous electrochemical capacitors. In addition to electrical double layer capacitance, redox reactions in this device contribute to charge storage at both positive and negative electrodes via a catholyte of IOx-/I- couple and a redox couple of H2O/Had, respectively. Here, we, for the first time, report utilizing IOx-/I- redox couple for the positive electrode, which pins the positive electrode potential to be 0.4-0.5 V vs Ag/AgCl. With the positive electrode potential pinned, we can polarize the cell to 1.6 V without breaking down the aqueous electrolyte so that the negative electrode potential could reach -1.1 V vs Ag/AgCl in the basic electrolyte, greatly enhancing energy storage. Both mass spectroscopy and Raman spectrometry confirm the formation of IO3- ions (+5) from I- (-1) after charging. Based on the total mass of electrodes and electrolyte in a practically relevant cell configuration, the device exhibits a maximum specific energy of 7.1 Wh/kg, operates between -20 and 50 °C, provides a maximum specific power of 6222 W/kg, and has a stable cycling life with 93% retention of the peak specific energy after 14,000 cycles.

  3. Determination of electron affinity of carbonyl radicals by means of negative ion mass spectrometry.

    PubMed

    Muftakhov; Vasil'ev; Mazunov

    1999-06-01

    Appearance energies of [M-H](-) ions from carbonyl compounds R-CO-R' (R,R' = H, CH(3), NH(2), OH) have been measured by means of negative ion mass spectrometry in resonant electron capture mode. Values of electron affinity of the corresponding radicals, CH(2)&dbond;C(X)O, NH&dbond;C(X)O and O&dbond;C(X)O, have been determined. Copyright 1999 John Wiley & Sons, Ltd. PMID:10407285

  4. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  5. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  6. Ligand Affinities Estimated by Quantum Chemical Calculations.

    PubMed

    Söderhjelm, Pär; Kongsted, Jacob; Ryde, Ulf

    2010-05-11

    We present quantum chemical estimates of ligand-binding affinities performed, for the first time, at a level of theory for which there is a hope that dispersion and polarization effects are properly accounted for (MP2/cc-pVTZ) and at the same time effects of solvation, entropy, and sampling are included. We have studied the binding of seven biotin analogues to the avidin tetramer. The calculations have been performed by the recently developed PMISP approach (polarizable multipole interactions with supermolecular pairs), which treats electrostatic interactions by multipoles up to quadrupoles, induction by anisotropic polarizabilities, and nonclassical interactions (dispersion, exchange repulsion, etc.) by explicit quantum chemical calculations, using a fragmentation approach, except for long-range interactions that are treated by standard molecular-mechanics Lennard-Jones terms. In order to include effects of sampling, 10 snapshots from a molecular dynamics simulation are studied for each biotin analogue. Solvation energies are estimated by the polarized continuum model (PCM), coupled to the multipole-polarizability model. Entropy effects are estimated from vibrational frequencies, calculated at the molecular mechanics level. We encounter several problems, not previously discussed, illustrating that we are first to apply such a method. For example, the PCM model is, in the present implementation, questionable for large molecules, owing to the use of a surface definition that gives numerous small cavities in a protein. PMID:26615702

  7. Affinity of guanosine derivatives for polycytidylate revisited

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Hurley, T. B.; Baird, E. E.

    1995-01-01

    Evidence is presented for complexation of guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG) with polycytidylate (poly(C)) at pH 8.0 and 23 degrees C in the presence of 1.0 M NaCl2 and 0.2 M MgCl2 in water. The association of 2-MeImpG with poly(C) was investigated using UV-vis spectroscopy as well as by monitoring the kinetics of the nucleophilic substitution reaction of the imidazole moiety by amines. The results of both methods are consistent with moderately strong poly(C) 2-MeImpG complexation and the spectrophotometric measurements allowed the construction of a binding isotherm with a concentration of 2-MeImpG equal to 5.55 +/- 0.15 mM at half occupancy. UV spectroscopy was employed to establish the binding of other guanosine derivatives on poly(C). These derivatives are guanosine 5'-monophosphate (5'GMP), guanosine 5'-monophosphate imidazolide (ImpG), and guanosine 5'-monophosphate morpholidate (morpG). Within experimental error these guanosine derivatives exhibit the same affinity for poly(C) as 2-MeImpG.

  8. Prostate Cancer and Bone: The Elective Affinities

    PubMed Central

    2014-01-01

    The onset of metastases dramatically changes the prognosis of prostate cancer patients, determining increased morbidity and a drastic fall in survival expectancy. Bone is a common site of metastases in few types of cancer, and it represents the most frequent metastatic site in prostate cancer. Of note, the prevalence of tumor relapse to the bone appears to be increasing over the years, likely due to a longer overall survival of prostate cancer patients. Bone tropism represents an intriguing challenge for researchers also because the preference of prostate cancer cells for the bone is the result of a sequential series of targetable molecular events. Many factors have been associated with the peculiar ability of prostate cancer cells to migrate in bone marrow and to determine mixed osteoblastic/osteolytic lesions. As anticipated by the success of current targeted therapy aimed to block bone resorption, a better understanding of molecular affinity between prostate cancer and bone microenvironment will permit us to cure bone metastasis and to improve prognosis of prostate cancer patients. PMID:24971315

  9. Banach frames in the affine synthesis problem

    NASA Astrophysics Data System (ADS)

    Terekhin, Pavel A.

    2009-10-01

    We consider the problem of representing functions f\\in L^p(\\mathbb R^d) by a series in elements of the affine system \\displaystyle \\psi_{j,k}(x)=\\lvert\\det a_j\\rvert^{1/2}\\psi(a_jx-bk), \\qquad j\\in\\mathbb N, \\quad k\\in\\mathbb Z^d. The corresponding representation theorems are established on the basis of the frame inequalities \\displaystyle A\\Vert g\\Vert _q\\le\\Vert\\{(g,\\psi_{j,k})\\}\\Vert _Y\\le B\\Vert g\\Vert _q for the Fourier coefficients \\displaystyle(g,\\psi_{j,k})=\\int_{\\mathbb R^d}g(x)\\psi_{j,k}(x)\\,dx of functions g\\in L^q(\\mathbb R^d), 1/p+1/q=1, where {\\Vert\\cdot\\Vert}_Y is the norm in some Banach space of number families \\{y_{j,k}\\} and 0 are constants. In particular, it is proved that if the integral of a function \\psi\\in L^1\\cap L^p(\\mathbb R^d), 1, is nonzero, so \\displaystyle\\int_{\\mathbb R^d}\\psi(x)\\,dx\

  10. Evaluation of p16/Ki-67 dual staining in detection of cervical precancer and cancers: a multicenter study in China

    PubMed Central

    Yu, Lu-Lu; Chen, Wen; Lei, Xiao-Qin; Qin, Yu; Wu, Ze-Ni; Pan, Qin-Jing; Zhang, Xun; Chang, Bai-Feng; Zhang, Shao-Kai; Guo, Hui-Qin; Qiao, You-Lin

    2016-01-01

    Purpose To analyze the clinical performance of p16/Ki-67 dual-stained cytology identifying high-grade cervical intraepithelial neoplasia (CIN2+) in Chinese women. Methods 1079 women attending ongoing cervical cancer screening and 211 “enriched” women aged ≥30yrs with biopsy-confirmed CIN2+ from five Chinese hospitals were enrolled during year 2014-2015. Cervical specimens were collected for high-risk human papillomavirus (HR-HPV) DNA analysis, Liquid-based cytology (LBC) and p16/Ki-67 dual staining. Colposcopy and biopsy were performed on women with any abnormal result. Results p16/Ki-67 positivity increased with histologic severity. It was 18.4%(183/996) in normal histology, 54.0%(34/63) in CIN1, 81.0%(34/42) in CIN2, 93.3%(111/119) in CIN3, 71.4% (5/7) in adenocarcinoma and 95.2%(60/63) in squamous cell carcinoma. Compared with the HR-HPV negatives, p16/Ki-67 expression was significantly higher in the HPV16/18 positive (OR: 35.45(95%CI: 23.35-53.84)) and other 12 HR-HPV types positive group (OR: 8.01(95%CI: 5.81-11.05). The sensitivity and specificity of p16/Ki-67 to detect CIN2+ in the entire population were 90.9% and 79.5%, respectively. In women with ASC-US and LSIL, sensitivity and specificity for detection of CIN2+ were 87.5% and 66.4%, respectively, with a referral rate of 43.8%. In women who tested positive for HR-HPV, sensitivity and specificity of dual-staining for detection of CIN2+ were 92.7% and 52.7%, respectively, and the referral rate was 68.7%. Conclusions p16/Ki-67 dual-stained cytology provided a high sensitivity and moderate specificity to detect underlying cervical precancer and cancers in various settings, and might be considered as an efficient screening tool in China. PMID:27029033

  11. CSAR Data Set Release 2012: Ligands, Affinities, Complexes, and Docking Decoys

    PubMed Central

    2013-01-01

    A major goal in drug design is the improvement of computational methods for docking and scoring. The Community Structure Activity Resource (CSAR) has collected several data sets from industry and added in-house data sets that may be used for this purpose (www.csardock.org). CSAR has currently obtained data from Abbott, GlaxoSmithKline, and Vertex and is working on obtaining data from several others. Combined with our in-house projects, we are providing a data set consisting of 6 protein targets, 647 compounds with biological affinities, and 82 crystal structures. Multiple congeneric series are available for several targets with a few representative crystal structures of each of the series. These series generally contain a few inactive compounds, usually not available in the literature, to provide an upper bound to the affinity range. The affinity ranges are typically 3–4 orders of magnitude per series. For our in-house projects, we have had compounds synthesized for biological testing. Affinities were measured by Thermofluor, Octet RED, and isothermal titration calorimetry for the most soluble. This allows the direct comparison of the biological affinities for those compounds, providing a measure of the variance in the experimental affinity. It appears that there can be considerable variance in the absolute value of the affinity, making the prediction of the absolute value ill-defined. However, the relative rankings within the methods are much better, and this fits with the observation that predicting relative ranking is a more tractable problem computationally. For those in-house compounds, we also have measured the following physical properties: logD, logP, thermodynamic solubility, and pKa. This data set also provides a substantial decoy set for each target consisting of diverse conformations covering the entire active site for all of the 58 CSAR-quality crystal structures. The CSAR data sets (CSAR-NRC HiQ and the 2012 release) provide substantial, publically

  12. The P 2 ' residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity

    SciTech Connect

    Salameh, Moh'd A.; Soares, Alexei S.; Hockla, Alexandra; Radisky, Derek C.; Radisky, Evette S.

    2011-11-15

    PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P2' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P1 and P2' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant Ki of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.

  13. Binding affinities and thermodynamics of noncovalent functionalization of carbon nanotubes with surfactants.

    PubMed

    Oh, Hyunkyu; Sim, Jinsook; Ju, Sang-Yong

    2013-09-01

    Binding affinity and thermodynamic understanding between a surfactant and carbon nanotube is essential to develop various carbon nanotube applications. Flavin mononucleotide-wrapped carbon nanotubes showing a large redshift in optical signature were utilized to determine the binding affinity and related thermodynamic parameters of 12 different nanotube chiralities upon exchange with other surfactants. Determined from the midpoint of sigmoidal transition, the equilibrium constant (K), which is inversely proportional to the binding affinity of the initial surfactant-carbon nanotube, provided quantitative binding strengths of surfactants as SDBS > SC ≈ FMN > SDS, irrespective of electronic types of SWNTs. Binding affinity of metallic tubes is weaker than that of semiconducting tubes. The complex K patterns from semiconducting tubes show preference to certain SWNT chiralities and surfactant-specific cooperativity according to nanotube chirality. Controlling temperature was effective to modulate K values by 30% and enables us to probe thermodynamic parameters. Equally signed enthalpy and entropy changes produce Gibbs energy changes with a magnitude of a few kJ/mol. A greater negative Gibbs energy upon exchange of surfactant produces an enhanced nanotube photoluminescence, implying the importance of understanding thermodynamics for designing nanotube separation and supramolecular assembly of surfactant.

  14. QSAR modeling of globulin binding affinity of corticosteroids using AM1 calculations.

    PubMed

    De, Kakali; Sengupta, Chandana; Roy, Kunal

    2004-06-15

    A quantitative structure-activity analysis of binding affinity of a series of 30 steroids for corticosteroid-binding globulin was performed using Wang-Ford charges of the non-hydrogen common atoms obtained from molecular electrostatic potential surface of AM1 optimized energy-minimized geometries of the compounds. Attempts were made to include lipophilicity (logP) and molar refractivity (MR) values of the whole molecules in the multivariate relations. The final relations were subjected to 'leave-one-out' cross-validation to check their predictive potential. It was found from the study that the charges of different atoms of the steroid nucleus [atoms 3, 4, 5 (ring A), 8, 9 (fusion points of rings B and C) and 16 (ring D)] contribute significantly to the binding affinity. This suggests the importance of these atoms/sites for the globulin binding affinity, which is also supported by previous reports on structure-activity relations of corticosteroids. Further, molar refractivity shows parabolic relation with the binding affinity, which indicates the possibility of dispersion interactions. The statistical qualities of the final equations generated in the present study (predicted variance 77-82%; explained variance 83-87%) are better than those of some of the previously reported models.

  15. Chemiluminescently labeled aptamers as the affinity probe for interaction analysis by capillary electrophoresis.

    PubMed

    Li, Hong-Yi; Deng, Qin-Pei; Zhang, De-Wen; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2010-07-01

    Aptamers are nucleic acid oligonucleotides, which can recognize targets with high affinity and specificity. Fluorescently labeled aptamers have been used as affinity probes in CE for interaction analysis. In this study, a method of labeling aptamers chemiluminescently with isoluminol isothiocyanate (ILITC) through covalent bonds was proposed and realized. The ILITC-labeled aptamers were characterized by HPLC-MS and purified by HPLC. After desalination, the ILITC-labeled aptamers were employed as the affinity probe for interaction analysis in CE coupled with chemiluminescence detection (CE-CL) by interface of end column reaction mode, the apparatus of which was home-designed and setup. CE-CL experiment conditions, including buffer pH, concentrations of horseradish peroxidase and H(2)O(2), were optimized first. The system of thrombin and its 29-mer aptamer was chosen as the model. Binding parameters, namely the dissociation constant (K(d)) and the binding site number (n), were calculated. The K(d) obtained was 124.0+/-6.9 nM in agreement with the reported values. Thus, interaction analysis method based on chemiluminescently labeled aptamers as the affinity probe in CE-CL has been established. This method can be widely applied due to the ease and universality of the labeling method, simplicity of CE-CL apparatus and combination with aptamers for a wide range of targets.

  16. Rationally manipulating aptamer binding affinities in a stem-loop molecular beacon.

    PubMed

    Armstrong, Rachel E; Strouse, Geoffrey F

    2014-10-15

    Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM - 203 μM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the Kd is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the ΔG values indicate a clear correlation between the aptamer hybridized length in the stem and its observed Kd. The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate Kd's for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes.

  17. Fluorotrimethylsilane affinities of anionic nucleophiles: a study of fluoride-induced desilylation.

    PubMed

    Krouse, Ian H; Wenthold, Paul G

    2005-05-01

    In this study, preparation and decomposition of five novel pentavalent fluorosiliconates, RSi(CH3)3F- (R = CH3CH2O, CF3CH2O, (CH3)2CHO, (CH3)3SiO, and (CH3)3SiNH) is used to investigate the process of fluoride-induced desilylation. The siliconates were characterized by collision-induced dissociation and energy-resolved mass spectrometry. Decomposition of RSi(CH3)3F- leads to loss of the nucleophile R- and FSi(CH3)3, except in the case of (CH3)3SiNHSi(CH3)3F-, where HF loss is also observed. Ion affinities for FSi(CH3)3 have been measured for all five nucleophiles, and compare well with computational predictions. The observed trend of the bond dissociation energies resembles the trend of deltaH(acid) values for the corresponding conjugate acids, RH. Additionally, this data has been incorporated with existing thermochemistry to derive fluoride affinities for four of the silanes (R = CH3CH2O, (CH3)2CHO, (CH3)3SiO, and (CH3)3SiNH). We use the fluoride affinity of the silanes and the FSi(CH3)3 affinity of the departing nucleophilic anion to assess the feasibility of fluoride-induced desilylation of the silanes examined in this work. PMID:15862771

  18. The low-affinity complex of cytochrome c and its peroxidase

    PubMed Central

    Van de Water, Karen; Sterckx, Yann G. J.; Volkov, Alexander N.

    2015-01-01

    The complex of yeast cytochrome c peroxidase and cytochrome c is a paradigm of the biological electron transfer (ET). Building on seven decades of research, two different models have been proposed to explain its functional redox activity. One postulates that the intermolecular ET occurs only in the dominant, high-affinity protein–protein orientation, while the other posits formation of an additional, low-affinity complex, which is much more active than the dominant one. Unlike the high-affinity interaction—extensively studied by X-ray crystallography and NMR spectroscopy—until now the binding of cytochrome c to the low-affinity site has not been observed directly, but inferred mainly from kinetics experiments. Here we report the structure of this elusive, weak protein complex and show that it consists of a dominant, inactive bound species and an ensemble of minor, ET-competent protein–protein orientations, which summarily account for the experimentally determined value of the ET rate constant. PMID:25944250

  19. Striving for Empathy: Affinities, Alliances and Peer Sexuality Educators

    ERIC Educational Resources Information Center

    Fields, Jessica; Copp, Martha

    2015-01-01

    Peer sexuality educators' accounts of their work reveal two approaches to empathy with their students: affinity and alliance. "Affinity-based empathy" rests on the idea that the more commonalities sexuality educators and students share (or perceive they share), the more they will be able to empathise with one another, while…

  20. Affine group formulation of the Standard Model coupled to gravity

    SciTech Connect

    Chou, Ching-Yi; Ita, Eyo; Soo, Chopin

    2014-04-15

    In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of the Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.

  1. Tending to Change: Toward a Situated Model of Affinity Spaces

    ERIC Educational Resources Information Center

    Bommarito, Dan

    2014-01-01

    The concept of affinity spaces, a theoretical construct used to analyze literate activity from a spatial perspective, has gained popularity among scholars of literacy studies and, particularly, video-game studies. This article seeks to expand current notions of affinity spaces by identifying key assumptions that have limited researchers'…

  2. High affinity retinoic acid receptor antagonists: analogs of AGN 193109.

    PubMed

    Johnson, A T; Wang, L; Gillett, S J; Chandraratna, R A

    1999-02-22

    A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated.

  3. Steric effects on alkyl cation affinities of maingroup-element hydrides.

    PubMed

    Ruiz, Juan M; Mulder, R Joshua; Fonseca Guerra, Célia; Bickelhaupt, F Matthias

    2011-03-01

    We have carried out an extensive exploration of gas-phase alkyl cation affinities (ACA) of archetypal anionic and neutral bases across the periodic system using zeroth order regular approximation-relativistic density functional theory at BP86/QZ4P//BP86/TZ2P. ACA values were computed for the methyl, ethyl, i-propyl and t-butyl cations and compared with the corresponding proton affinities (PA). One purpose of this work is to provide an intrinsically consistent set of values of the 298 K ACA of all anionic (XH (n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (group 18) along the periods 1-6. Another purpose is to determine and rationalize the trend in affinity for a cation as the latter varies from proton to t-butyl cation. This undertaking is supported by quantitative bond energy decomposition analyses. Correlations are established between PA and ACA values. PMID:20882538

  4. Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

    PubMed Central

    Veldhuis, Gertjan; Broos, Jaap; Poolman, Bert; Scheek, Ruud M.

    2005-01-01

    Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeIImtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a low-affinity binding site with KD-values in the nano- and micromolar range, respectively. However, reported KD-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the discrepancies reported in literature, regarding both the variability in KD-values and the binding stoichiometry. By comparing the binding capacity obtained with flow dialysis with different methods to determine the protein concentration (UV-protein absorption, Bradford protein detection, and a LDH-linked protein assay to quantify the number of phosphorylation sites), we proved the existence of only one mannitol binding site per dimeric species of unphosphorylated EnzymeIImtl. Furthermore, the affinity of EnzymeIImtl for mannitol appeared to be dependent on the protein concentration and seemed to reflect the presence of an endogenous ligand. The dependency could be simulated assuming that >50% of the binding sites were occupied with a ligand that shows an affinity for EnzymeIImtl in the same range as mannitol. PMID:15879478

  5. Probes for narcotic receptor mediated phenomena. 43. Synthesis of the ortho-a and para-a, and improved synthesis and optical resolution of the ortho-b and para–b oxide-bridged phenylmorphans: Compounds with moderate to low opioid-receptor affinity

    PubMed Central

    Li, Feng; Folk, John E.; Cheng, Kejun; Kurimura, Muneaki; Deck, Jason A.; Deschamps, Jeffrey R.; Rothman, Richard B.; Dersch, Christina M.; Jacobson, Arthur E.; Rice, Kenner C.

    2011-01-01

    N-Phenethyl-substituted ortho-a and para-a oxide-bridged phenylmorphans have been obtained through an improved synthesis and their binding affinity examined at the various opioid receptors. Although the N-phenethyl substituent showed much greater affinity for μ- and κ-opioid receptors than their N-methyl relatives (e.g., Ki = 167 nM and 171 nM at μ- and κ-receptors vs >2800 and 7500 nM for the N-methyl ortho-a oxide-bridged phenylmorphan), the a-isomers were not examined further because of their relatively low affinity. The N-phenethyl substituted ortho-b and para-b oxide-bridged phenylmorphans were also synthesized and their enantiomers were obtained using supercritical fluid chromatography. Of the four enantiomers, only the (+)-ortho-b isomer had moderate affinity for μ- and κ-receptors (Ki = 49 and 42 nM, respectively, and it was found to also have moderate μ- and κ-opioid antagonist activity in the [35S]GTP-γ-S assay (Ke = 31 and 26 nM). PMID:21684752

  6. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  7. Detection of protein-protein interactions using tandem affinity purification.

    PubMed

    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

  8. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  9. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture. PMID:27473483

  10. In vivo dynamics and kinetics of pKi-67: Transition from a mobile to an immobile form at the onset of anaphase

    SciTech Connect

    Saiwaki, Takuya; Kotera, Ippei; Sasaki, Mitsuho; Takagi, Masatoshi; Yoneda, Yoshihiro . E-mail: yyoneda@anat3.med.osaka-u.ac.jp

    2005-08-01

    A cell proliferation marker protein, pKi-67, distributes to the chromosome periphery during mitosis and nucleolar heterochromatin in the interphase. We report here on the structural domains of pKi-67 that are required for its correct distribution. While both the LR domain and the conserved domain were involved in localization to the nucleolar heterochromatin, both the LR domain and the Ki-67 repeat domain were required for its distribution to the mitotic chromosome periphery. Using in vivo time-lapse microscopy, GFP-pKi-67 was dynamically tracked from the mitotic chromosome periphery to reforming nucleoli via prenucleolar bodies (PNBs). The signals in PNBs then moved towards and fused into the reforming nucleoli with a thin string-like fluorescence during early G1 phase. An analysis of the in vivo kinetics of pKi-67 using photobleaching indicated that the association of pKi-67 with chromatin was progressively altered from 'loose' to 'tight' after the onset of anaphase. These findings indicate that pKi-67 dynamically alters the nature of the interaction with chromatin structure during the cell cycle, which is closely related to the reformation process of the interphase nucleolar chromatin.

  11. Chasing polys: Interdisciplinary affinity and its connection to physics identity

    NASA Astrophysics Data System (ADS)

    Scott, Tyler D.

    This research is based on two motivations that merge by means of the frameworks of interdisciplinary affinity and physics identity. First, a goal of education is to develop interdisciplinary abilities in students' thinking and work. But an often ignored factor is students interests and beliefs about being interdisciplinary. Thus, this work develops and uses a framework called interdisciplinary affinity. It encompasses students interests in making connections across disciplines and their beliefs about their abilities to make those connections. The second motivation of this research is to better understand how to engage more students with physics. Physics identity describes how a student sees themselves in relation to physics. By understanding how physics identity is developed, researchers and educators can identify factors that increase interest and engagement in physics classrooms. Therefore, physics identity was used in conjunction with interdisciplinary affinity. Using a mixed methods approach, this research used quantitative data to identify the relationships interdisciplinary affinity has with physics identity and the physics classroom. These connections were explored in more detail using a case study of three students in a high school physics class. Results showed significant and positive relationships between interdisciplinary affinity and physics identity, including the individual interest and recognition components of identity. It also identified characteristics of physics classrooms that had a significant, positive relationship with interdisciplinary affinity. The qualitative case study highlighted the importance of student interest to the relationship between interdisciplinary affinity and physics identity. It also identified interest and mastery orientation as key to understanding the link between interdisciplinary affinity and the physics classroom. These results are a positive sign that by understanding interdisciplinary affinity and physics identity

  12. Ki67 score as a potential predictor in the selection of liver-directed therapies for metastatic neuroendocrine tumors: a single institutional experience

    PubMed Central

    Singla, Smit; LeVea, Charles M.; Pokuri, Venkata K.; Attwood, Kristopher M.; Wach, Michael M.; Tomaszewski, Garin M.; Kuvshinoff, Boris

    2016-01-01

    Background Neuroendocrine tumors (NETs) metastatic to the liver are treated with transarterial radioembolization (TARE) using yttrium-90 (Y-90) microspheres or transarterial chemoembolization (TACE). However the criteria for patient selection are not well defined. We sought to determine if Ki67 score could help select patients for one therapy over the other in the management of hepatic neuroendocrine metastases. Methods Single institution analysis of patients treated with Y-90 or TACE between 2001 and 2014. Pathologists blinded to clinical information performed Ki67 staining. Data were analyzed using multivariate association for survival outcomes. Results Amongst 72 patients (male: 39, female: 33, median age: 57 years) with metastatic NET, the most common site of origin was small bowel (n=35, 49%), while pancreas constituted 32% (n=23). Forty-four patients were treated with Y-90 (61%) and 28 patients received TACE (39%). Ki67 score was available in 28 patients (64%) treated with Y-90 and 16 patients (57%) with TACE. Within Y-90 group, there was greater use of Sandostatin (95% vs. 75%, P=0.02) and less number of total treatments completed (89% vs. 46%, P<0.001). There was no significant difference in overall survival (OS) between Y-90 and TACE when used without selection (median, 69 vs. 82 months, P=0.47). When adjusted for Ki67, patients with Ki67 score ≥3% had better OS with Y-90 compared to TACE (HR, 0.1; CI, 0.01–0.9), however for Ki67 <3%, OS was better when treated with TACE compared to Y-90 (HR, 13.5; CI, 1.22–148.87). Conclusions There is significant interaction between Ki-67 score and liver-directed treatment benefit in patients with hepatic neuroendocrine metastases. Ki-67 score ≥3% predicts greater benefit with Y-90 and a Ki-67 score <3% predicts greater benefit with TACE. PMID:27284478

  13. Enzyme-gold affinity labelling of cellulose.

    PubMed

    Berg, R H; Erdos, G W; Gritzali, M; Brown, R D

    1988-04-01

    The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Optimal affine-invariant matching: performance characterization

    NASA Astrophysics Data System (ADS)

    Costa, Mauro S.; Haralick, Robert M.; Shapiro, Linda G.

    1992-04-01

    The geometric hashing scheme proposed by Lamdan and Wolfson can be very efficient in a model-based matching system, not only in terms of the computational complexity involved, but also in terms of the simplicity of the method. In a recent paper, we discussed errors that can occur with this method due to quantization, stability, symmetry, and noise problems. These errors make the original geometric hashing technique unsuitable for use on the factory floor. Beginning with an explicit noise model, which the original Lamdan and Wolfson technique lacks, we derived an optimal approach that overcomes these problems. We showed that the results obtained with the new algorithm are clearly better than the results from the original method. This paper addresses the performance characterization of the geometric hashing technique, more specifically the affine-invariant point matching, applied to the problem of recognizing and determining the pose of sheet metal parts. The experiments indicate that with a model having 10 to 14 points, with 2 points of the model undetected and 10 extraneous points detected, and with the model points perturbed by Gaussian noise of standard deviation 3 (0.58 of range), the average amount of computation required to obtain an answer is equivalent to trying 11 of the possible three-point bases. The misdetection rate, measured by the percentage of correct bases matches that fail to verify, is 0.9. The percentage of incorrect bases that successfully produced a match that did verify (false alarm rate) is 13. And, finally, 2 of the experiments failed to find a correct match and verify it. Results for experiments with real images are also presented.

  15. Correlation between the Uptake of 18F-Fluorodeoxyglucose (18F-FDG) and the Expression of Proliferation-Associated Antigen Ki-67 in Cancer Patients: A Meta-Analysis

    PubMed Central

    Deng, Sheng-ming; Zhang, Wei; Zhang, Bin; Chen, Yin-yin; Li, Ji-hui; Wu, Yi-wei

    2015-01-01

    , esophageal and colorectal cancers, and poor in head and neck, thyroid, gastric and malignant melanoma tumors. Subgroup analysis indicated that positron emission tomography (PET) or PET/CT imaging technology or Ki-67 and standardized uptake value (SUV) measurement technology did not significantly affect the results of r values, and Begg's test showed no significant publication bias. Conclusion In cancer patients, 18F-FDG uptake showed a moderate positive correlation with tumor cell proliferation. Different tumor types exhibited varied degree of correlation, and the correlation was significant in TETs and GSTs. However, our results need further validation by clinical trials with a large sample of different tumor types. PMID:26038827

  16. Association of Angiopoietin-2 and Ki-67 Expression with Vascular Density and Sunitinib Response in Metastatic Renal Cell Carcinoma

    PubMed Central

    Mirtti, Tuomas; Ristimäki, Ari; Joensuu, Heikki; Bono, Petri; Saharinen, Pipsa

    2016-01-01

    The Angiopoietin-2 (Ang2, Angpt2) growth factor is a context-dependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase and known to promote tumour angiogenesis and metastasis. Angiopoietin antagonists have been tested in clinical cancer trials in combination with VEGF-based anti-angiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. Here, we evaluated, using immunohistochemistry, the expression of Ang2, CD31 and the cell proliferation marker Ki-67 in the primary kidney cancer from 136 mRCC patients, who received first-line sunitinib after nephrectomy. Ang2 protein expression was restrained to RCC tumour vessels, and correlated with tumour vascularization and response to sunitinib. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS, 15.7 vs. 28.5 months, P = 0.015). In summary, in this study to investigate endothelial Ang2 in mRCC patients treated with first-line sunitinib, high cancer Ang2 expression was associated with the CBR, but not PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS. PMID:27100185

  17. The KiSS-1/GPR54 system: putative target for endocrine disruption of reproduction at hypothalamic-pituitary unit?

    PubMed

    Navarro, Victor M; Tena-Sempere, Manuel

    2008-04-01

    The hypothalamic-pituitary (HP) unit, key element in the control of development and function of the reproductive axis, is highly sensitive to the organizing and activational effects of endogenous and exogenous compounds with sex steroid activity. Thus, inappropriate sex steroid input during early critical periods of their maturation can induce immediate or delayed defects in the neuroendocrine systems governing reproduction, which might eventually lead to alterations in the timing of puberty onset and/or infertility. Similarly, later inadequate exposures can cause dysfunctions of the gonadotropic axis. In recent years, kisspeptins (the products of KiSS-1 gene that operate through the G protein-coupled receptor 54) have emerged as fundamental regulators of puberty onset and gonadotropin secretion, and neurons in the hypothalamus expressing KiSS-1 have been demonstrated as essential conduits for the dynamic control of the gonadotropic axis by a number of hormonal factors. In this context, the hypothalamic KiSS-1 system has been proven sensitive to the early organizing effects, as well as to the acute regulatory actions, of gonadal steroids: phenomena which are likely to play fundamental roles in the sexual differentiation of gonadotropin secretion, and its feedback regulation by androgen and oestrogen. These observations raise the question on whether the hypothalamic KiSS-1 system might constitute a potential target for endocrine disruption of puberty onset and reproductive function at the HP unit by compounds with oestrogenic, androgenic or anti-androgenic activity. We review herein the physiological basis and initial, albeit so far scarce, experimental evidence supporting such possibility.

  18. KI and WU Polyomaviruses and CD4+ Cell Counts in HIV-1–infected Patients, Italy

    PubMed Central

    Babakir-Mina, Muhammed; Ciccozzi, Massimo; Farchi, Francesca; Bergallo, Massimiliano; Cavallo, Rossana; Adorno, Gaspare; Perno, Carlo Federico

    2010-01-01

    To investigate an association between KI and WU polyomavirus (KIPyV and WUPyV) infections and CD4+ cell counts, we tested HIV-1–positive patients and blood donors. No association was found between cell counts and virus infections in HIV-1–positive patients. Frequency of KIPyV infection was similar for both groups. WUPyV was more frequent in HIV-1–positive patients. PMID:20735940

  19. Selectively Promiscuous Opioid Ligands: Discovery of High Affinity/Low Efficacy Opioid Ligands with Substantial Nociceptin Opioid Peptide Receptor Affinity

    PubMed Central

    2015-01-01

    Emerging clinical and preclinical evidence suggests that a compound displaying high affinity for μ, κ, and δ opioid (MOP, KOP, and DOP) receptors and antagonist activity at each, coupled with moderate affinity and efficacy at nociceptin opioid peptide (NOP) receptors will have utility as a relapse prevention agent for multiple types of drug abuse. Members of the orvinol family of opioid ligands have the desired affinity profile but have typically displayed substantial efficacy at MOP and or KOP receptors. In this study it is shown that a phenyl ring analogue (1d) of buprenorphine displays the desired profile in vitro with high, nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo. PMID:24761755

  20. Limited proteolysis for assaying ligand binding affinities of nuclear receptors.

    PubMed

    Benkoussa, M; Nominé, B; Mouchon, A; Lefebvre, B; Bernardon, J M; Formstecher, P; Lefebvre, P

    1997-01-01

    The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

  1. Proliferative activity (ki-67 expression) and outcome in high grade osteosarcoma: a study of 27 cases.

    PubMed

    Jong, R; Davis, A M; Mendes, M G; Wunder, J S; Bell, R S; Kandel, R

    2000-01-01

    Purpose. Although pre-operative chemotherapy has improved the prognosis for individuals with osteosarcoma, approximately 40% of patients will die of their disease.The aim of this study was to quantitate proliferative activity in high grade osteosarcomas and to determine whether proliferation is a prognostic factor.Patients. The study consisted of 27 patients with high grade non-metastatic osteosarcoma at various sites for whom pre-operative biopsies and resection specimens were available for review. All patients were treated similarly and had at least 24 months' follow-up from the date of diagnosis.Methods. Proliferative activity (Ki-67 expression) was examined in the diagnostic biopsies immunohistochemically using the MIB-1 antibody. Proliferation was quantitated in two ways; (1) the number of immunopositive cells was counted manually using an ocular grid; or (2) the percentage of immunopositive nuclear area was assessed using morphometric image analysis. Proliferative index was evaluated in relation to patient outcome.Results. Proliferative activity was seen in all biopsies.The median proliferative index as determined by counting cells was 24% (mean of 27%, range of 7-61%) and by image analysis was 2% (mean 3%, range 0.32-8.4).The correlation between MIB-1 proliferation indices determined either by image analysis methodology or manual cell counting was high (Spearman's rho=0.79). Proliferative index did not appear to predict either disease-free or overall survival.Discussion. Tumor proliferation does not appear to be prognostic for high grade osteosarcomas.Whether assessment of this feature in conjunction with other tumor characteristics might be prognostic requires further study. PMID:18521434

  2. CD147 and Ki-67 overexpression confers poor prognosis in squamous cell carcinoma of oral tongue: A tissue microarray study

    PubMed Central

    Yu, Yau-Hua; Morales, Jose; Feng, Lei; Lee, J. Jack; El-Naggar, Adel K.; Vigneswaran, Nadarajah

    2015-01-01

    Squamous cell carcinoma of the oral tongue (SCCOT) exhibits high risk for recurrence and regional metastasis even after surgical resection. We assessed the clinicopathologic and prognostic significance of a group of functionally related biomarkers. We used a tissue microarray consisting SCCOT from 32 patients for this study. These patients were treated at the UT- M.D. Anderson Cancer Center from 1995 to 2008. Biomarker expression levels were examined by immunohistochemistry and graded semiquantitatively to determine their prognostic significance. CD147 and Tp63 expressions were significantly associated with a higher T-stage and Ki-67 labelling index as well as shorter overall survival (OS). Expression of Tp63 associated positively with poorly-differentiated histology. There was significant association of Tp63 with the expression levels of CD147 and Glut-1. Glut-1 overexpression was marginally associated with a higher T-stage. There was no prognostic significance of CD44v6 expression in SCCOT. SCCOT with CD147 overexpression in combination with high Ki-67 labelling index had poor OS. CD147 and Ki-67 overexpression is associated with aggressive disease with poor prognosis in SCCOT. PMID:25747176

  3. KiSThelP: a program to predict thermodynamic properties and rate constants from quantum chemistry results.

    PubMed

    Canneaux, Sébastien; Bohr, Frédéric; Henon, Eric

    2014-01-01

    Kinetic and Statistical Thermodynamical Package (KiSThelP) is a cross-platform free open-source program developed to estimate molecular and reaction properties from electronic structure data. To date, three computational chemistry software formats are supported (Gaussian, GAMESS, and NWChem). Some key features are: gas-phase molecular thermodynamic properties (offering hindered rotor treatment), thermal equilibrium constants, transition state theory rate coefficients (transition state theory (TST), variational transition state theory (VTST)) including one-dimensional (1D) tunnelling effects (Wigner, and Eckart) and Rice-Ramsperger-Kassel-Marcus (RRKM) rate constants, for elementary reactions with well-defined barriers. KiSThelP is intended as a working tool both for the general public and also for more expert users. It provides graphical front-end capabilities designed to facilitate calculations and interpreting results. KiSThelP enables to change input data and simulation parameters directly through the graphical user interface and to visually probe how it affects results. Users can access results in the form of graphs and tables. The graphical tool offers customizing of 2D plots, exporting images and data files. These features make this program also well-suited to support and enhance students learning and can serve as a very attractive courseware, taking the teaching content directly from results in molecular and kinetic modelling. PMID:24190715

  4. Probes for narcotic receptor mediated phenomena. 44. Synthesis of an N-substituted 4-hydroxy-5-(3-hydroxyphenyl)morphan with high affinity and selective μ-antagonist activity

    PubMed Central

    Iyer, Malliga R.; Lee, Yong Sok; Deschamps, Jeffrey R.; Dersch, Christina M.; Rothman, Richard B.; Jacobson, Arthur E.; Rice, Kenner C.

    2012-01-01

    A simple three-step synthesis of 5-(3-hydroxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonan-4-ol (3a) was achieved using an osmium tetroxide mediated oxidation of the known intermediate 6. A pyrrolidine-ring variant of 3a (3-(7-(hydroxymethyl)-6-methyl-6-azabicyclo[3.2.1]octan-1-yl)phenol (5)) was isolated when other routes were used. The epimeric hydroxy analogue 4a was synthesized by simple inversion of the stereochemistry at C-4. Both N-methyl (3a and 4a) and N-phenethyl (3b and 4b) derivatives were synthesized. The compounds were examined for their opioid receptor affinity and the N-phenethyl analogue 3b was found to have relatively weak affinity for the μ-opioid receptor (Ki = 74 nM). However, the N-phenethyl analogue of the C-4 epimer, 4b, had about 15 fold higher affinity than 3b and was selective for the μ-opioid receptor (Ki = 4.6 nM). Compound 4b was a moderately potent μ-opioid antagonist (Ke = 12 nM), as determined by [35S]GTP-γ-S assays. Compounds 3b and 4b were energy minimized at the level of B3LYP/6-31G*, and then overlaid onto the 5-phenylmorphan, the (1R,5R,9S)-(−)-enantiomer of 2b (Fig. 1) with the α or β-OH group at the C-9 position. The spatial orientation of the hydroxyl moiety in 3b, 4b, 2a, and 2b is proposed to be the structural requirement for high μ-opioid receptor binding affinity and their agonist or antagonist activity. The modest change in spatial position of the hydroxyl moiety, and not the N-substituent, induced the change from potent agonist to an antagonist of moderate potency. PMID:22341895

  5. Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

    PubMed

    Zhao, Wei-Wei; Liu, Fu-Feng; Shi, Qing-Hong; Sun, Yan

    2014-09-12

    In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations. PMID:25064536

  6. Proliferative potential in benign mixed salivary gland tumors and its value in primary and recurrent neoplasms.

    PubMed

    Kazanceva, Anna; Groma, Valerie; Smane, Liene; Kornevs, Egils; Teibe, Uldis

    2011-01-01

    OBJECTIVE. Mixed salivary gland tumors are characterized by a marked diversity in the cell proliferation. It course in the stromal component, and, especially in recurrent neoplasms, is not completely understood. This study evaluated cell proliferative potential, its value and the clinical course of primary and recurrent salivary gland pleomorphic adenomas (PA). MATERIALS AND METHODS. 322 benign salivary gland tumors were used in this study. The cell proliferation was estimated by Ki-67 expression levels. RESULTS. Ki-67 immunoreactivity showed a wide range of spectra; in the epithelial and stromal type of PA the cell proliferation had the value from 0.07±0.03 (95% CI 0.01-0.14) to 4.81±0.60 (95% CI 3.61-6.02) and from 0 to 0.79±0.11 (95% CI 0.57-1.00), respectively. The Ki-67 value was higher in recurrent tumors compared with primary, and the mean number of Ki-67-positive cells per visual microscopic field constituted 2.14±1.60 (95% CI 1.47-2.47) comparing with 1.43 (95% CI 0.97-1.55) revealed in primary tumors. CONCLUSION. Cell proliferation values correlate with a recurrence of neoplasm, and elevation of proliferation potential in the stromal component of recurrent PA is indicative of clinical course change for the worse.

  7. High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice

    PubMed Central

    2015-01-01

    The dopamine D3 receptor (D3R) is a promising target for the development of pharmacotherapeutics to treat substance use disorders. Several D3R-selective antagonists are effective in animal models of drug abuse, especially in models of relapse. Nevertheless, poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in translating these drug candidates to clinical use. Herein, we report a series of D3R-selective 4-phenylpiperazines with improved metabolic stability. A subset of these compounds was evaluated for D3R functional efficacy and off-target binding at selected 5-HT receptor subtypes, where significant overlap in SAR with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin in wild-type but not D3R knockout mice. PMID:26203768

  8. How Structure Defines Affinity in Protein-Protein Interactions

    PubMed Central

    Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

    2014-01-01

    Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

  9. Analysis of biomolecular interactions using affinity microcolumns: a review.

    PubMed

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L; White, Christopher J; Carter, NaTasha; Hage, David S

    2014-10-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  10. Value Added?

    ERIC Educational Resources Information Center

    UCLA IDEA, 2012

    2012-01-01

    Value added measures (VAM) uses changes in student test scores to determine how much "value" an individual teacher has "added" to student growth during the school year. Some policymakers, school districts, and educational advocates have applauded VAM as a straightforward measure of teacher effectiveness: the better a teacher, the better students…

  11. Affine differential geometry analysis of human arm movements.

    PubMed

    Flash, Tamar; Handzel, Amir A

    2007-06-01

    Humans interact with their environment through sensory information and motor actions. These interactions may be understood via the underlying geometry of both perception and action. While the motor space is typically considered by default to be Euclidean, persistent behavioral observations point to a different underlying geometric structure. These observed regularities include the "two-thirds power law", which connects path curvature with velocity, and "local isochrony", which prescribes the relation between movement time and its extent. Starting with these empirical observations, we have developed a mathematical framework based on differential geometry, Lie group theory and Cartan's moving frame method for the analysis of human hand trajectories. We also use this method to identify possible motion primitives, i.e., elementary building blocks from which more complicated movements are constructed. We show that a natural geometric description of continuous repetitive hand trajectories is not Euclidean but equi-affine. Specifically, equi-affine velocity is piecewise constant along movement segments, and movement execution time for a given segment is proportional to its equi-affine arc-length. Using this mathematical framework, we then analyze experimentally recorded drawing movements. To examine movement segmentation and classification, the two fundamental equi-affine differential invariants-equi-affine arc-length and curvature are calculated for the recorded movements. We also discuss the possible role of conic sections, i.e., curves with constant equi-affine curvature, as motor primitives and focus in more detail on parabolas, the equi-affine geodesics. Finally, we explore possible schemes for the internal neural coding of motor commands by showing that the equi-affine framework is compatible with the common model of population coding of the hand velocity vector when combined with a simple assumption on its dynamics. We then discuss several alternative explanations

  12. Diacylglycerol kinase η1 is a high affinity isozyme for diacylglycerol.

    PubMed

    Komenoi, Suguru; Takemura, Fumika; Sakai, Hiromichi; Sakane, Fumio

    2015-05-01

    Diacylglycerol kinase (DGK) η plays important roles in various patho-physiological events such as oncogenesis. In this study, we performed an enzymological characterization of DGKη splice variant 1 (DGKη1). The Km value for diacylglycerol was 0.14 mol%. Intriguingly, the Km value of DGKη1 for diacylglycerol was at least 9-fold lower than those of other DGK isozymes including DGKα, indicating that DGKη1 is a high affinity isozyme for diacylglycerol. Therefore, DGKη1 is a unique DGK isozyme, which may function at particular membrane sites where only low concentrations of diacylglycerol are supplied.

  13. Affinity- and topology-dependent bound on current fluctuations

    NASA Astrophysics Data System (ADS)

    Pietzonka, Patrick; Barato, Andre C.; Seifert, Udo

    2016-08-01

    We provide a proof of a recently conjectured universal bound on current fluctuations in Markovian processes. This bound establishes a link between the fluctuations of an individual observable current, the cycle affinities driving the system into a non-equilibrium steady state, and the topology of the network. The proof is based on a decomposition of the network into independent cycles with both positive affinity and positive stationary cycle current. This formalism allows for a refinement of the bound for systems in equilibrium or with locally vanishing affinities.

  14. Affinity+: Semi-Structured Brainstorming on Large Displays

    SciTech Connect

    Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.; LaMothe, Ryan R.; Endert, Alexander

    2013-04-27

    Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

  15. ANALYSIS OF DRUG INTERACTIONS WITH HIGH DENSITY LIPOPROTEIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Sike; Sobansky, Matthew R.; Hage, David S.

    2009-01-01

    Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography. This approach was evaluated by using it to examine the binding of high density lipoprotein (HDL) to the drugs propranolol or verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPLC columns with reproducible binding to propranolol over four to five days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interactions were occurring between R/S-propranolol and HDL at 37°C: saturable binding with an association equilibrium constant (Ka) of 1.1–1.9 × 105 M−1, and non-saturable binding with an overall affinity constant (n Ka) of 3.7–4.1 × 104 M−1. Similar results were found at 4 and 27°C. Verapamil also gave similar behavior, with a Ka of 6.0 × 104 M−1 at 37°C for the saturable sites and a n Ka value for the non-saturable sites of 2.5 × 104 M−1. These measured affinities gave good agreement with solution-phase values. The results indicated HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body. PMID:19833090

  16. Theoretical and Experimental Determination of the Proton Affinity of (CF3CH2)2O

    NASA Technical Reports Server (NTRS)

    Zehe, Michael J.; Ball, David W.

    1998-01-01

    We report the experimental determination of the proton affinity of the molecule (CF3CH2)2O using chemical ionization mass spectrometry, and we compare it to the theoretical value obtained for protonation at the oxygen atom using the calculational methodology (MP2/6-31G**//MP2/3-21G). The proton affinity for this molecule as measured by bracketing experiments was between 724 kJ/mole and 741 kJ/mole. Ab initio (MP2/6-31G**//MP2/3-21G) calculations yield a value of about 729 kJ/mole, in agreement with the chemical ionization experiments. The results of these and related calculations suggest that the (MP2/6-31G**//MP2/3-21G) methodology is acceptable for estimating the proton affinities of partially-and fully-fluorinated methyl and ethyl ethers. We submit that any conclusions about the chemistry of fluoroether polymer lubricants based on their basicity can also be predicted reliably with such calculations.

  17. Pre-Yield Non-Affine Fluctuations and A Hidden Critical Point in Strained Crystals

    PubMed Central

    Das, Tamoghna; Ganguly, Saswati; Sengupta, Surajit; Rao, Madan

    2015-01-01

    A crystalline solid exhibits thermally induced localised non-affine droplets in the absence of external stress. Here we show that upon an imposed shear, the size of these droplets grow until they percolate at a critical strain, well below the value at which the solid begins to yield. This critical point does not manifest in most thermodynamic or mechanical properties, but is hidden and reveals itself in the onset of inhomogeneities in elastic moduli, marked changes in the appearance and local properties of non-affine droplets and a sudden enhancement in defect pair concentration. Slow relaxation of stress and an-elasticity appear as observable dynamical consequences of this hidden criticality. Our results may be directly verified in colloidal crystals with video microscopy techniques but are expected to have more general validity. PMID:26039380

  18. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M.

  19. Electron affinities of d1 transition metal chloride clusters and onset of super halogen behavior

    NASA Astrophysics Data System (ADS)

    Behera, Swayamprabha; Joseph, Jorly; Jena, Purusottam

    2011-03-01

    Geometry, electronic structure, and electron affinity of d1 transition metal chloride clusters (MCl n , M = Sc,Y, La; n = 1--5) have been calculated using density functional theory. Chlorine atoms are chemically bound in all cases except for MCl 5 . The electron affinities of MCl n (n = 1--3) are small and increase only marginally as a function of n until the valence of the metal atom is consumed. Beyond this, they rise sharply and reach a value of 5.96, 6.03 and 5.90 eV for ScCl 4 , YCl 4 and LaCl 4 , respectively and remain high for n = 5. MCl n , (n = 4,5) clusters, therefore, behave as superhalogens. Results are compared with available experimental data

  20. Electron affinities of d1 transition metal chloride clusters and onset of super halogen behavior

    NASA Astrophysics Data System (ADS)

    Joseph, Jorly; Behera, Swayamprabha; Jena, Purusottam

    2010-09-01

    Geometry, electronic structure, and electron affinity of d1 transition metal chloride clusters (MCl n, M = Sc, Y, La; n = 1-5) have been calculated using density functional theory. Chlorine atoms are chemically bound in all cases except for MCl 5. The electron affinities of MCl n ( n = 1-3) are small and increase only marginally as a function of n until the valence of the metal atom is consumed. Beyond this, they rise sharply and reach a value of 5.96, 6.03 and 5.90 eV for ScCl 4, YCl 4 and LaCl 4, respectively and remain high for n = 5. MCl n, ( n = 4,5) clusters, therefore, behave as superhalogens. Results are compared with available experimental data.

  1. Self-Affinity, Self-Similarity and Disturbance of Soil Seed Banks by Tillage.

    PubMed

    Dias, Luís S

    2013-07-05

    Soil seed banks were sampled in undisturbed soil and after soil had been disturbed by tillage (tine, harrow or plough). Seeds were sorted by size and shape, and counted. Size-number distributions were fitted by power law equations that allowed the identification of self-similarity and self-affinity. Self-affinity and thus non-random size-number distribution prevailed in undisturbed soil. Self-similarity and thus randomness of size-number distribution prevailed after tillage regardless of the intensity of disturbance imposed by cultivation. The values of fractal dimensions before and after tillage were low, suggesting that short-term, short-range factors govern size-number distribution of soil seed banks.

  2. Superconformal Chern-Simons partition functions of affine D-type quiver from Fermi gas

    NASA Astrophysics Data System (ADS)

    Moriyama, Sanefumi; Nosaka, Tomoki

    2015-09-01

    We consider the partition function of the superconformal Chern-Simons theories with the quiver diagram being the affine D-type Dynkin diagram. Rewriting the partition function into that of a Fermi gas system, we show that the perturbative expansions in 1 /N are summed up to an Airy function, as in the ABJM theory or more generally the theories of the affine A-type quiver. As a corollary, this provides a proof for the previous proposal in the large N limit. For special values of the Chern-Simons levels, we further identify three species of the membrane instantons and also conjecture an exact expression of the overall constant, which corresponds to the constant map in the topological string theory. [Figure not available: see fulltext.

  3. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M. PMID:23325100

  4. Method for resurrecting negative electron affinity photocathodes after exposure to an oxidizing gas

    DOEpatents

    Mulhollan, Gregory A; Bierman, John C

    2012-10-30

    A method by which negative electron affinity photocathodes (201), single crystal, amorphous, or otherwise ordered, can be made to recover their quantum yield following exposure to an oxidizing gas has been discovered. Conventional recovery methods employ the use of cesium as a positive acting agent (104). In the improved recovery method, an electron beam (205), sufficiently energetic to generate a secondary electron cloud (207), is applied to the photocathode in need of recovery. The energetic beam, through the high secondary electron yield of the negative electron affinity surface (203), creates sufficient numbers of low energy electrons which act on the reduced-yield surface so as to negate the effects of absorbed oxidizing atoms thereby recovering the quantum yield to a pre-decay value.

  5. Enhanced starch hydrolysis using α-amylase immobilized on cellulose ultrafiltration affinity membrane.

    PubMed

    Konovalova, Viktoriia; Guzikevich, Kateryna; Burban, Anatoliy; Kujawski, Wojciech; Jarzynka, Karolina; Kujawa, Joanna

    2016-11-01

    In order to prepare ultrafiltration membranes possessing biocatalytic properties, α-amylase has been immobilized on cellulose membranes. Enzyme immobilization was based on a covalent bonding between chitosan and a surface of cellulose membrane, followed by an attachment of Cibacron Blue F3G-A dye as affinity ligand. Various factors affecting the immobilization process, such as enzyme concentration, pH of modifying solution, zeta-potential of membrane surface, and stability of immobilized enzyme were studied. The applicability of immobilized α-amylase has been investigated in ultrafiltration processes. The immobilization of α-amylase on membrane surface allows to increase the value of mass transfer coefficient and to decrease the concentration polarization effect during ultrafiltration of starch solutions. The enzyme layer on the membrane surface prevents a rapid increase of starch concentration due to the amylase hydrolysis of starch in the boundary layer. The presented affinity immobilization technique allows also for the regeneration of membranes from inactivated enzyme.

  6. Community Development, Transitional Value, and Institutional Affinity: Outdoor Orientation Program Impacts

    ERIC Educational Resources Information Center

    Howard, Ryan A.; O'Connell, Timothy S.; Lathrop, Anna H.

    2016-01-01

    This article examines the impact of an outdoor orientation program (OOP) on a cohort of first-year university students who participated in a canoe trip facilitated by peer leaders. The curriculum included training for outdoor skills and transitional guidance to university life (i.e., strategies for time management, critical thinking, becoming…

  7. Antigen retrieval using pH 3.5 glycine-HCl buffer or urea solution for immunohistochemical localization of Ki-67.

    PubMed

    Shi, S R; Chaiwun, B; Young, L; Imam, A; Cote, R J; Taylor, C R

    1994-07-01

    A new antibody (MIB-1) has been described, permitting the demonstration of Ki-67 proliferation antigen in paraffin sections. However, satisfactory results were obtained only after subjecting tissue sections to microwave based antigen retrieval in citrate buffer solution. Other buffer solutions produce equivalent or better results and also permit use of the original Ki-67 antibody, which hitherto has been considered ineffective for paraffin sections.

  8. Dense Stereo Matching Method Based on Local Affine Model.

    PubMed

    Li, Jie; Shi, Wenxuan; Deng, Dexiang; Jia, Wenyan; Sun, Mingui

    2013-07-01

    A new method for constructing an accurate disparity space image and performing an efficient cost aggregation in stereo matching based on local affine model is proposed in this paper. The key algorithm includes a new self-adapting dissimilarity measurement used for calculating the matching cost and a local affine model used in cost aggregation stage. Different from the traditional region-based methods, which try to change the matching window size or to calculate an adaptive weight to do the aggregation, the proposed method focuses on obtaining the efficient and accurate local affine model to aggregate the cost volume while preserving the disparity discontinuity. Moreover, the local affine model can be extended to the color space. Experimental results demonstrate that the proposed method is able to provide subpixel precision disparity maps compared with some state-of-the-art stereo matching methods. PMID:24163727

  9. On the thermodynamic basis of the affinity decay rate

    NASA Astrophysics Data System (ADS)

    Garcia-Colín, L. S.; Piña, E.; de la Selva, S. M. T.

    1990-03-01

    In the past five years exhaustive studies in chemical reactions have lead to an empirical equation describing how isothermal-isometric homogeneous reactions evolve towards equilibrium independently of their particular mechanism or rate law. Such an equation expresses the time rate of change of the chemical affinity as a linear function of the inverse of time. In this paper we show that by invoking the local equilibrium hypothesis one may provide, a time evolution equation for the chemical affinity that is uniquely given by the solution of the particular rate law of the reaction considered. Consequently such an equation is not of the same functional form for all reactions. On the other hand, integration of Dalton's law under specific initial conditions, together with the local equilibrium assumption and the ideality requirement for the reacting species, exhibits a unique inverse time decay for the chemical affinity. This explains the good fitting of the inverse in time dependence of the chemical affinity with experimental data.

  10. A thermodynamic approach to the affinity optimization of drug candidates.

    PubMed

    Freire, Ernesto

    2009-11-01

    High throughput screening and other techniques commonly used to identify lead candidates for drug development usually yield compounds with binding affinities to their intended targets in the mid-micromolar range. The affinity of these molecules needs to be improved by several orders of magnitude before they become viable drug candidates. Traditionally, this task has been accomplished by establishing structure activity relationships to guide chemical modifications and improve the binding affinity of the compounds. As the binding affinity is a function of two quantities, the binding enthalpy and the binding entropy, it is evident that a more efficient optimization would be accomplished if both quantities were considered and improved simultaneously. Here, an optimization algorithm based upon enthalpic and entropic information generated by Isothermal Titration Calorimetry is presented.

  11. Frontal affinity chromatography (FAC): theory and basic aspects.

    PubMed

    Kasai, Ken-ichi

    2014-01-01

    Frontal affinity chromatography (FAC) is a versatile analytical tool for determining specific interactions between biomolecules and is particularly useful in the field of glycobiology. This article presents its basic aspects, merits, and theory. PMID:25117240

  12. Bidirectional Elastic Image Registration Using B-Spline Affine Transformation

    PubMed Central

    Gu, Suicheng; Meng, Xin; Sciurba, Frank C.; Wang, Chen; Kaminski, Naftali; Pu, Jiantao

    2014-01-01

    A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-Spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bi-directional instead of the traditional unidirectional objective / cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy. PMID:24530210

  13. Antibody Affinity Maturation in Fishes—Our Current Understanding

    PubMed Central

    Magor, Brad G.

    2015-01-01

    It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes. PMID:26264036

  14. Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines.

    PubMed

    Cozzi, Nicholas V; Daley, Paul F

    2016-02-01

    correlations among physicochemical properties of the congeneric 5-substituted-DALT compounds. The descriptors included electronic (σp), hydrophobic (π), and steric (CMR) parameters. The binding affinity at 5-HT1A, 5-HT1D, 5-HT7, and κ opioid receptors was positively correlated with the steric volume parameter CMR. At α2A, α2B, and α2C receptors, and at the histamine H1 receptor, binding affinity was correlated with the Hammett substituent parameter σp; higher affinity was associated with larger σp values. At the σ2 receptor, higher affinity was correlated with increasing π. These correlations should aid in the development of more potent and selective drugs within this family of compounds.

  15. Hydride affinities of cumulated, isolated, and conjugated dienes in acetonitrile.

    PubMed

    Zhu, Xiao-Qing; Liang, Hao; Zhu, Yan; Cheng, Jin-Pei

    2008-11-01

    The hydride affinities (defined as the enthalpy changes in this work) of 15 polarized dienes [five phenyl sulfone substituted allenes (1a), the corresponding five isolated dienes (1b), and the corresponding five conjugated dienes (1c)] in acetonitrile solution were determined by titration calorimetry for the first time. The results display that the hydride affinity scales of the 15 dienes in acetonitrile range from -71.6 to -73.9 kcal/mol for 1a, from -46.2 to -49.7 kcal/mol for 1b, and from -45.0 to -46.5 kcal/mol for 1c, which indicates that the hydride-obtaining abilities of the cumulated dienes (1a) are not only much larger than those of the corresponding conjugated dienes (1c) but also much larger than those of the corresponding isolated dienes (1b). The hydrogen affinities of the 15 dienes as well as the hydrogen affinities and the proton affinities of the radical anions of the dienes (1(-*)) in acetonitrile were also evaluated by using relative thermodynamic cycles according to Hess's law. The results show that (i) the hydrogen affinities of the neutral dienes 1 cover a range from -44.5 to -45.6 kcal/mol for 1a, from -20.4 to -21.4 kcal/mol for 1b, and from -17.3 to -18.5 kcal/mol for 1c; (ii) the hydrogen affinities of the radical anions of the dienes (1(-*)) in acetonitrile cover a range from -40.6 to -47.2 kcal/mol for 1a(-*), from -21.6 to -29.6 kcal/mol for 1b(-*), and from -10.0 to -15.4 kcal/mol for 1c(-*); (iii) the proton affinities of the 15 1a(-*) in acetonitrile cover a range from -97.0 to -100.6 kcal/mol for 1a(-*), from -77.8 to -83.4 kcal/mol for 1b(-*), and from -66.2 to -68.9 kcal/mol for 1c(-*). The main reasons for the great difference between the cumulated dienes and the corresponding isolated and conjugated dienes in the hydride affinity, hydrogen affinity, and proton affinity have been examined. It is evident that these experimental results should be quite valuable to facilitate the elucidation of the origins of the especially high

  16. The stellar-to-halo mass relation of GAMA galaxies from 100 deg2 of KiDS weak lensing data

    NASA Astrophysics Data System (ADS)

    van Uitert, Edo; Cacciato, Marcello; Hoekstra, Henk; Brouwer, Margot; Sifón, Cristóbal; Viola, Massimo; Baldry, Ivan; Bland-Hawthorn, Joss; Brough, Sarah; Brown, M. J. I.; Choi, Ami; Driver, Simon P.; Erben, Thomas; Heymans, Catherine; Hildebrandt, Hendrik; Joachimi, Benjamin; Kuijken, Konrad; Liske, Jochen; Loveday, Jon; McFarland, John; Miller, Lance; Nakajima, Reiko; Peacock, John; Radovich, Mario; Robotham, A. S. G.; Schneider, Peter; Sikkema, Gert; Taylor, Edward N.; Verdoes Kleijn, Gijs

    2016-07-01

    We study the stellar-to-halo mass relation of central galaxies in the range 9.7 < log 10(M*/h- 2 M⊙) < 11.7 and z < 0.4, obtained from a combined analysis of the Kilo Degree Survey (KiDS) and the Galaxy And Mass Assembly (GAMA) survey. We use ˜100 deg2 of KiDS data to study the lensing signal around galaxies for which spectroscopic redshifts and stellar masses were determined by GAMA. We show that lensing alone results in poor constraints on the stellar-to-halo mass relation due to a degeneracy between the satellite fraction and the halo mass, which is lifted when we simultaneously fit the stellar mass function. At M* > 5 × 1010 h- 2 M⊙, the stellar mass increases with halo mass as {˜ }M_h^{0.25}. The ratio of dark matter to stellar mass has a minimum at a halo mass of 8 × 1011 h-1 M⊙ with a value of M_h/M_{*}=56_{-10}^{+16} [h]. We also use the GAMA group catalogue to select centrals and satellites in groups with five or more members, which trace regions in space where the local matter density is higher than average, and determine for the first time the stellar-to-halo mass relation in these denser environments. We find no significant differences compared to the relation from the full sample, which suggests that the stellar-to-halo mass relation does not vary strongly with local density. Furthermore, we find that the stellar-to-halo mass relation of central galaxies can also be obtained by modelling the lensing signal and stellar mass function of satellite galaxies only, which shows that the assumptions to model the satellite contribution in the halo model do not significantly bias the stellar-to-halo mass relation. Finally, we show that the combination of weak lensing with the stellar mass function can be used to test the purity of group catalogues.

  17. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    SciTech Connect

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-07-23

    Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  18. Comparison of the Manual, Semiautomatic, and Automatic Selection and Leveling of Hot Spots in Whole Slide Images for Ki-67 Quantification in Meningiomas

    PubMed Central

    Swiderska, Zaneta; Korzynska, Anna; Markiewicz, Tomasz; Lorent, Malgorzata; Zak, Jakub; Wesolowska, Anna; Roszkowiak, Lukasz; Slodkowska, Janina; Grala, Bartlomiej

    2015-01-01

    Background. This paper presents the study concerning hot-spot selection in the assessment of whole slide images of tissue sections collected from meningioma patients. The samples were immunohistochemically stained to determine the Ki-67/MIB-1 proliferation index used for prognosis and treatment planning. Objective. The observer performance was examined by comparing results of the proposed method of automatic hot-spot selection in whole slide images, results of traditional scoring under a microscope, and results of a pathologist's manual hot-spot selection. Methods. The results of scoring the Ki-67 index using optical scoring under a microscope, software for Ki-67 index quantification based on hot spots selected by two pathologists (resp., once and three times), and the same software but on hot spots selected by proposed automatic methods were compared using Kendall's tau-b statistics. Results. Results show intra- and interobserver agreement. The agreement between Ki-67 scoring with manual and automatic hot-spot selection is high, while agreement between Ki-67 index scoring results in whole slide images and traditional microscopic examination is lower. Conclusions. The agreement observed for the three scoring methods shows that automation of area selection is an effective tool in supporting physicians and in increasing the reliability of Ki-67 scoring in meningioma. PMID:26240787

  19. The Role of Neurotransmitters in Protection against Amyloid-β Toxicity by KiSS-1 Overexpression in SH-SY5Y Neurons

    PubMed Central

    Milton, Nathaniel G. N.

    2013-01-01

    Recent studies have suggested that the kisspeptin (KP) and kissorphin (KSO) peptides have neuroprotective actions against the Alzheimer's amyloid-β (Aβ) peptide. Overexpression of the human KiSS-1 gene that codes for KP and KSO peptides in SH-SY5Y neurons has also been shown to inhibit Aβ neurotoxicity. The in vivo actions of KP include activation of neuroendocrine and neurotransmitter systems. The present study used antagonists of KP, neuropeptide FF (NPFF), opioids, oxytocin, estrogen, adrenergic, cholinergic, dopaminergic, serotonergic, and γ-aminobutyric acid (GABA) receptors plus inhibitors of catalase, cyclooxygenase, nitric oxide synthase, and the mitogen activated protein kinase cascade to characterize the KiSS-1 gene overexpression neuroprotection against Aβ cell model. The results showed that KiSS-1 overexpression is neuroprotective against Aβ and the action appears to involve the KP or KSO peptide products of KiSS-1 processing. The mechanism of neuroprotection does not involve the activation of the KP or NPFF receptors. Opioids play a role in the toxicity of Aβ in the KiSS-1 overexpression system and opioid antagonists naloxone or naltrexone inhibited Aβ toxicity. The mechanism of KiSS-1 overexpression induced protection against Aβ appears to have an oxytocin plus a cyclooxygenase dependent component, with the oxytocin antagonist atosiban and the cyclooxygenase inhibitor SC-560 both enhancing the toxicity of Aβ. PMID:24967306

  20. Flexible Linker Modulates Glycosaminoglycan Affinity of Decorin Binding Protein A.

    PubMed

    Morgan, Ashli; Sepuru, Krishna Mohan; Feng, Wei; Rajarathnam, Krishna; Wang, Xu

    2015-08-18

    Decorin binding protein A (DBPA) is a glycosaminoglycan (GAG)-binding adhesin found on the surface of the bacterium Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease. DBPA facilitates bacterial adherence to extracellular matrices of human tissues and is crucial during the early stage of the infection process. Interestingly, DBPA from different strains (B31, N40, and PBr) show significant differences in GAG affinities, but the structural basis for the differences is not clear. In this study, we show that GAG affinity of N40 DBPA is modulated in part by flexible segments that control access to the GAG binding site, such that shortening of the linker leads to higher GAG affinity when analyzed using ELISA, gel mobility shift assay, solution NMR, and isothermal titration calorimetry. Our observation that GAG affinity differences among different B. burgdorferi strains can be attributed to a flexible linker domain regulating access to the GAG-binding domain is novel. It also provides a rare example of how neutral amino acids and dynamic segments in GAG binding proteins can have a large influence on GAG affinity and provides insights into why the number of basic amino acids in the GAG-binding site may not be the only factor determining GAG affinity of proteins. PMID:26223367

  1. Identification and affinity of very high affinity binding sites for the phenylalkylamine series of Ca/sup +/ channel blockers in the Drosophila nervous system

    SciTech Connect

    Pauron, D.; Qar, J.; Barhanin, J.; Fournier, D.; Cuany, A.; Pralavorio, M.; Berge, J.B.; Lazdunski, M.

    1987-10-06

    The interaction of putative Ca/sup 2 +/ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-(/sup 3/H)D888 and (+/-)-(/sup 3/H)verapamil. These ligands recognize a single class of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a K/sub d/ value as exceptional low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca/sup 2 +/ channel blockers as well as bepridil inhibited (-)-(/sup 3/H)D888 binding in a competitive way with K/sub d/ values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor was used in photoaffinity experiments. A protein of M/sub r/ 135,000 +/- 5000 was specifically labeled after ultraviolet irradiation.

  2. Synthesis and Characterization of [76Br]-Labeled High Affinity A3 Adenosine Receptor Ligands for Positron Emission Tomography

    PubMed Central

    Kiesewetter, Dale O.; Lang, Lixin; Ma, Ying; Bhattacharjee, Abesh Kumar; Gao, Zhan-Guo; Joshi, Bhalchandra V.; Melman, Artem; de Castro, Sonia; Jacobson, Kenneth A.

    2009-01-01

    Introduction Bromine-76 radiolabeled analogues of previously reported high affinity A3 adenosine receptor (A3AR) nucleoside ligands have been prepared as potential radiotracers for Positron Emission Tomography (PET). Methods The radiosyntheses were accomplished by oxidative radiobromination on the N6-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. Results We prepared an agonist ligand {[76Br](1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol (MRS3581)} in 59% radiochemical yield (RCY) with a specific activity of 19.5 GBq/μmol and an antagonist ligand {[76Br](1R,2R,3S,4R,5S)-4-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol. (MRS5147)} in 65% RCY with a specific activity of 22 GBq/μmol). The resultant products exhibited the expected high affinity (Ki ~ 0.6 nM) and specific binding at the human A3AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A3AR, over a 2 h time course, which suggests the presence of a specific A3AR retention mechanism. Conclusion We were able to compare uptake of the [76Br]labeled antagonist MRS5147 to [76Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A3AR rich tissue, suggesting that this ligand may have promise as a molecular imaging agent. PMID:19181263

  3. CHARACTERIZATION OF THE BINDING OF SULFONYLUREA DRUGS TO HSA BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Joseph, K.S.; Hage, David S.

    2010-01-01

    Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (± 0.2) × 105 M−1 and 3.5 (± 3.0) × 102 M−1 for acetohexamide and values of 8.7 (± 0.6) × 104 and 8.1 (± 1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (± 0.1) × 105 and 4.3 (± 0.3) × 104 M−1, respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (± 0.2) × 104 and 5.3 (± 0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions. PMID:20435530

  4. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics.

    PubMed

    Aweda, Tolulope A; Meares, Claude F

    2012-02-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.

  5. Quantitative structure-activity relationships for polychlorinated hydroxybiphenyl estrogen receptor binding affinity: An assessment of conformer flexibility

    SciTech Connect

    Bradbury, S.P.; Ankley, G.T.; Mekenyan, O.G.

    1996-11-01

    A diverse group of xenobiotics has a high binding affinity to the estrogen receptor (ER), suggesting that it can accommodate large variability in ligand structure. Relationships between xenobiotic surface, binding affinity, and estrogenic response have been suggested to be dependent on the conformational structures of the ligands. To explore the influence of conformational flexibility on ER binding affinity, a quantitative structure-activity relationship (QSAR) study was undertaken with estradiol, diethylstilbestrol, and a set of polychlorinated hydroxybiphenyls (PCHBs) of environmental concern. Although the low-energy minima of the PCHB congeners suggested that interconversions among conformers were likely, the electronic parameters associated with the conformer geometries for a specific PCHB congener could vary significantly. The results of the QSAR analysis suggested that among the PCHBs studied, the most polarizable conformers (lower absolute volume polarizability values) were most closely associated with ER binding affinity. Across the set of polarizable conformers, which did not include the low-energy gas-phase conformers, the electron donating properties of the hydroxy moiety and the aromatic component of the estradiol A ring analogue in the PCHBs were found to be correlated with higher ER binding affinity.

  6. Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

    SciTech Connect

    Jagadeesh, G.; Deth, R.C.

    1987-11-01

    Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of (/sup 3/H)prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate (Gpp(NH)p). A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. (/sup 3/H)Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with (/sup 3/H)prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.

  7. Temporal variation in phenetic affinity of early Upper Egyptian male cranial series.

    PubMed

    Keita, S O Y; Boyce, A J

    2008-04-01

    We carried out an exploratory historical biology study using temporally distinguished groups of predynastic-Early Dynastic male crania from the region of Upper Egypt. The objectives were, first, to determine the overall pattern of phenetic affinity between temporally sequential series and in relation to the earliest series and, second, to explore the possible meanings of the pattern of relationship to sociohistorical change. The cranial series were designated early predynastic, late predynastic, terminal predynastic, and Dynasty I. Craniometric phenetic affinity was ascertained using Mahalanobis distances; a 5% level of probability was chosen for significance. The distance matrix values were ordered into hierarchies of dissimilarity from each series (distance hierarchies) and tabulated for time-successive groups, including the temporally earliest series (i.e., serialized by time). The principal observations were as follows. The overall pattern was not one in which the values between all series were statistically insignificant; nor was it one of consistent sequential increase of biological distance from the earliest series. There was a notable and statistically significant distance between the early and late predynastic groups, with the late and terminal predynastic groups mutually having the lowest and statistically insignificant distances with each other. The value between the terminal predynastic and Dynasty I series was generally larger than the values between other groups and was statistically significant. The overall pattern is possibly consistent with archeological interpretations that postulate increasing intraregional interactions during the late and terminal predynastic periods and the rise of an Egyptian state that eventually included northern Egypt. PMID:18720900

  8. Analysis of Aged Human Serum Albumin Affinity for Doxazosin.

    PubMed

    Chudzik, Mariola; Równicka-Zubik, Joanna; Pożycka, Jadwiga; Pawelczak, Bartosz; Sulkowska, Anna

    2016-01-01

    Structural changes of human serum albumin (HSA) caused by old age and coexisting diseases result in differences in the binding of doxazosin (DOX). DOX is a postsynaptic α1- adrenoreceptor antagonist used for treatment of hypertension and benign prostatic hyperplasia. In elderly people suffering from various renal or hepatic diseases the significant portion of N-form of human serum albumin (normal) is converted to A-form (aged). The differences in binding of doxazosin to N- and Aform of albumin are an important factor, which may determines therapeutic dosage and toxicity of the test drug. To indicate these differences, the technique of fluorescence spectroscopy was used. The association constant (Ka) obtained from fluorescence quenching demonstrated that doxazosin has higher affinity for AHSA than for HSA. In order to describe the cooperativity in binding process, the values of the Hill's coefficient has been analysed. For DOX-HSA system (λex 295 nm) Hill's coefficient is close to 1 and it indicates that there is a single class of binding sites. For DOX-HSA (λex 275 nm) and DOX-AHSA (λex 275 nm and λex 295 nm) systems we observed positive cooperativity (nH>1). A greater red shift of fluorescence emission maximum of AHSA than HSA in the presence of DOX was observed. This suggests that the binding of DOX to AHSA was accompanied by a stronger increase in polarity around the fluorophores in comparison to HSA. The binding interaction between DOX and HSA has been also studied by molecular docking simulation.

  9. Microvascular density and endothelial area correlate with Ki-67 proliferative index in surgically-treated pancreatic ductal adenocarcinoma patients

    PubMed Central

    AMMENDOLA, MICHELE; SACCO, ROSARIO; MARECH, ILARIA; SAMMARCO, GIUSEPPE; ZUCCALÀ, VALERIA; LUPOSELLA, MARIA; PATRUNO, ROSA; GIORDANO, MARCELLA; RUGGIERI, EUSTACHIO; ZIZZO, NICOLA; GADALETA, COSMO DAMIANO; RANIERI, GIROLAMO

    2015-01-01

    Previous experimental and clinical data have indicated that tumour cell proliferation is associated with angiogenesis; in addition, an increased microvascular density (MVD) of tumours has been associated with poor prognosis in solid and haematological malignancies. However, limited data exists regarding the association between tumour cell proliferation and angiogenesis in primary tumour tissue from pancreatic ductal adenocarcinoma (PDAC) patients; therefore, the present study aimed to investigate this association. A series of 31 PDAC patients with stage Tumour (T)2–3 Node (N)0–1 Metastasis (M)0 were recruited into the present study and subsequently underwent surgery. PDAC tissue and adjacent normal tissue (ANT), resected during surgery, were evaluated using immunohistochemistry and image analysis methods to determine MVD, endothelial area (EA) and Ki-67 expression, which is an indicator of cell proliferation rate. The results demonstrated a correlation between the above parameters with each other as well as the main clinico-pathological features of PDAC. Significant differences were identified in MVD, EA and Ki-67 proliferation index between PDAC and ANT. It was demonstrated that MVD, EA and Ki-67 proliferation index were significantly correlated with each other in tumour tissue (r=0.69–0.81; P=0.001–0.003). However, no other significant correlations were identified. These data therefore suggested that angiogenesis and cell proliferation rate were significantly increased in PDAC compared with ANT, which provides a biological basis for the potential use of novel combinations of angiogenesis inhibitors and anti-proliferative chemotherapeutic drugs in the treatment of PDAC. PMID:26622606

  10. Is Ki-67 Expression Prognostic for Local Relapse in Early-Stage Breast Cancer Patients Treated With Breast Conservation Therapy (BCT)?

    SciTech Connect

    Hafeez, Farhaan; Neboori, Hanmanth J.; Harigopal, Malini; Wu, Hao; Haffty, Bruce G.; Yang, Qifeng; Schiff, Devora; Moran, Meena S.

    2013-10-01

    Purpose: Ki-67 is a human nuclear protein whose expression is strongly up-regulated in proliferating cells and can be used to determine the growth fraction in clonal cell populations. Although there are some data to suggest that Ki-67 overexpression may be prognostic for endpoints such as survival or postmastectomy recurrence, further elucidation of its prognostic significance is warranted. Specifically after breast conservation therapy (BCT) (defined in this setting as breast-conserving surgery and adjuvant radiation therapy), whether Ki-67 predicts for locoregional recurrence has not been investigated. The purpose of this study was to assess Ki-67 expression in a cohort of early-stage breast cancer patients to determine whether a significant independent association between Ki-67 and locoregional relapse exists. Methods and Materials: Ki-67 staining was conducted on a tissue microarray of 438 patients previously treated with BCT, and expression was analyzed with clinicopathologic features and outcomes from our database. Results: Ki-67 expression was more prevalent in black patients (37% of black patients vs 17% of white patients, P<.01), younger patients (27% of patients aged ≤50 years vs 15% of patients aged >50 years, P<.01), estrogen receptor (ER)–negative tumors (25% of ER-negative tumors vs 17% of ER-positive tumors, P=.04), human epidermal growth factor receptor 2 (HER2)/neu–positive tumors (35% of HER2-positive tumors vs 18% of HER2-negative tumors, P=.01), and larger tumors (26% of T2 tumors vs 16% of T1 tumors, P=.03). On univariate/multivariate analysis, Ki-67 did not predict for overall survival (74.4% vs 72.6%), cause-specific survival (82.9% vs 82.1%), local relapse-free survival (83.6% vs 88.5%), distant metastasis-free survival (76.1% vs 81.4%), recurrence-free survival (65.5% vs 74.6%), and locoregional recurrence-free survival (81.6% vs 84.7%): P>.05 for all. Conclusions: Ki-67 appears to be a surrogate marker for aggressive disease and

  11. Value Added

    ERIC Educational Resources Information Center

    Wilson, M. Roy

    2015-01-01

    With more than a thousand honors programs or colleges in the United States and that number growing every year, defining the value of honors is a significant undertaking. Honors seems to have become an obligatory upgrade that no college or university president can afford to be without, but there is more than institutional trending to be considered,…

  12. Value Added

    ERIC Educational Resources Information Center

    Welch, Matt

    2004-01-01

    This article profiles retiring values teacher Gene Doxey and describes his foundational contributions to the students of California's Ramona Unified School District. Every one of the Ramona Unified School District's 7,200 students is eventually funneled through Doxey's Contemporary Issues class, a required rite of passage between elementary school…

  13. Estimation of enthalpy data for reactions involving gas phase ions utilizing lattice potential energies: fluoride ion affinities (FIA) and pF- values of mSbF5(l) and mSbF5(g) (m = 1, 2, 3), AsF5(g), AsF5.SO2(c). Standard enthalpies of formation: Delta(f)H degrees (SbmF5m+1)(-),g) (m = 1, 2, 3), Delta(f)H degrees (AsF6(-),g), and Delta(f)H degrees (NF4+,g).

    PubMed

    Jenkins, H Donald Brooke; Roobottom, H K; Passmore, Jack

    2003-05-01

    Fluoride ion affinity (FIA) values (and the associated pF(-) values) are difficult to establish experimentally for pentafluorides of arsenic and antimony. Our approach, utilizing estimated lattice potential energies, provides a further opportunity to establish this data for liquid (and gaseous) SbF(5) and gaseous AsF(5) which compliments values obtained using ab initio routes for monomeric gas phase molecules and adds to results based on rigorous methods. A strategy is developed whereby construction of (multiple) Born-Fajans-Haber cycles centered around the (target) FIA reaction of interest yield a plethora of estimates for the enthalpy change of interest. This general approach is illustrated here by specific estimation of some experimentally based FIA values of SbF(5) and AsF(5). FIA values/kJ mol(-1) and pF- values estimated in this paper are FIA(SbF(5),l) approximately equal to -475 (+/-63), pF-(SbF(5),l) = 11.4 (+/-1.5); FIA(SbF(5),g) approximately equal to -506 (+/-63), pF-(SbF(5),g) = 12.4 (+/-1.5); FIA(2SbF(5),l) approximately equal to -609 (+/-63), pF- (2SbF(5),l) = 14.6 (+/-1.5); FIA (2SbF(5),g) approximately equal to -671 (+/-63), pF- (2SbF(5),g) = 16.0 (+/-1.5); FIA (3SbF(5),l) approximately -635 (+/-39), pF(-) (3SbF(5),l) = 15.2 (+/-0.9); FIA(3SbF(5),g) approximately -728 (+/-39), pF(-) (3SbF(5),g) = 17.4 (+/-0.9); FIA(AsF(5),g) approximately equal to -421 (+/-22), pF(-) (AsF(5),g) = 10.1 (+/- 0.5); and FIA (AsF(5).SO(2),s) approximately equal to -390 (+/-22), pF(-) (AsF(5).SO(2),s) = 9.3 (+/-0.5). Related standard enthalpies of formation (in kJ mol(-1)) are also assigned: Delta(f)H degrees (SbF(6)(-),g) approximately equal to -2075 (+/-52); Delta(f)H degrees (Sb(2)F(11)(-),g) approximately equal to -3520 (+/-63); Delta(f)H degrees (Sb(3)F(16)(-),g) approximately equal to -4874 (+/-39); Delta(f)H degrees (NF(4)(+),g) approximately equal to 903 (+/-32); Delta(f)H degrees (AsF(6)(-),g) approximately equal to -1907 (+/-22).

  14. Binding of ionic species: a general approach to measuring binding constants and assessing affinities.

    PubMed

    Roelens, Stefano; Vacca, Alberto; Venturi, Chiara

    2009-03-01

    Bound together: The association of receptors with ionic species cannot be assimilated to the binding of neutral guests. When dealing with salts, both ion pairing and binding to the free and the ion-paired ionic guest determine the actual association pattern (see figure). The general issue of measuring association constants and assessing affinities for ions is addressed and validated in two cases of anion binding.A general approach to the largely underestimated issue of measuring binding constants and assessing affinities in the binding of ionic species is described. The approach is based on a rigorous, nongraphical determination of binding constants in multiequilibrium systems by nonlinear regression of chemical shift data from NMR titrations and on the use of the BC(50) descriptor for assessing affinities and ranking the binding ability of receptors on a common scale. The approach has been validated with two tripodal anion-binding receptors, namely, a ureidic (1) and a pyrrolic (2) receptor, binding to tetramethylammonium chloride in CDCl(3)/CD(3)CN (80:20). A set of five and six formation constants could be measured for 1 and 2, respectively, including, in addition to the ion pair, complexes of the free and the ion-paired anion. The BC(50) values calculated from the measured constants allowed a quantitative assessment of each receptor's binding affinity towards the chloride anion, the pyrrolic receptor showing a 15-fold larger affinity over the ureidic receptor, a figure that quantifies the improvement obtained by replacing the amido-pyrrolic for ureidic binding groups on the tripodal scaffold of the receptor. The results have shown that, in contrast to common practice, neither of the two systems could be appropriately described by a 1:1 association with the anion only, but required the ion-pairing and ion-pair binding equilibria to be taken into account because these contribute substantially to the complexation process. The BC(50) descriptor has also been shown

  15. Erythrocyte 2,3-DPG, ATP and oxygen affinity in hemodialysis patients.

    PubMed

    Ninness, J R; Kimber, R W; McDonald, J W

    1974-10-01

    Patients on a chronic hemodialysis regimen were studied with respect to their erythrocyte adaptation to anemia. Erythrocyte 2,3-diphosphoglycerate (DPG) concentration was suboptimal compared with that of anemic patients who were not uremic. In uremic patients erythrocyte 2,3-DPG correlated poorly with hemoglobin level but more strongly with plasma pH. Differences between observed levels of erythrocyte 2,3-DPG and the values predicted using data from other anemic patients also correlated with pH. Gradual correction of plasma pH with oral sodium bicarbonate resulted in a substantial increase in erythrocyte 2,3-DPG and a decrease in oxygen affinity. Therefore, maintenance of normal pH in uremic subjects may improve tissue oxygenation. On the other hand, the rapid correction of acidosis during dialysis resulted in increased oxygen affinity. This response was due to the direct effect of pH on oxygen affinity in the absence of a significant change in erythrocyte 2,3-